Trehalose vaccine formulation

Abstract

Stable liquid, lyophilized, and frozen formulations are provided for live-attenuated and / or vector-based vaccines.

Description

TREHALOSE VACCINE FORMULATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 600,206, filed November 17, 2023, the contents of which are incorporated by reference herein in its entirety.CRADA STATEMENT

[0002] This invention was created in the performance of a Cooperative Research and Development Agreement with the National Institutes of Health, an Agency of the Department of Health and Human Services. The Government of the United States has certain rights in this invention.SEQUENCE LISTING

[0003] The present application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 2, 2024, is named “01121-0052-00PCT-ST26.xml” and is 42,875 bytes in size.BACKGROUND

[0004] Vaccines in liquid form are often unstable during storage and their instability can reduce their safety and efficacy, as well as increase cost and complexity of manufacture and storage. This is especially true for live-attenuated vaccines that are sensitive to changes in the environment. Vaccines are made up of proteins and other macromolecules that may change upon exposure to heat, light, oxygen, or radiation, or that may interact with the container materials or other components of the vaccine mixture. Determining these relationships and optimizing stability from production to administration to the patient is therefore an important part of vaccine development. There is a benefit to producing vaccines that are stable in liquid formulations that can be kept at room temperature, or at 2-8 °C, up until the moment they are used. Benefits include, for example, reducing the number of steps required when thawing or reconstituting a frozen or lyophilized vaccine, reducing manufacturing costs by allowing all manufacturing steps to be done in the liquid phase without a need to alter the virus form as compared to the form desired for administration, and allowing for more widespread distribution without the need for refrigeration. Moreover, there are time and cost savings in manufacturing and storing a vaccine that is liquid and shelf-stable at room or refrigerator temperatures so thatthe vaccine can be manufactured in pre-filled syringes or other forms for immediate administration to subjects, for example. Liquid formulations that allow for stability during holding steps during manufacturing and for stability for freeze-thaw steps are particularly desired. Liquid formulations can also be more easily administered to a subject immediately off the shelf without reconstitution, reducing human error in administration steps. The development of thermally stable vaccines can greatly alleviate this problem and, in turn, increase vaccine accessibility worldwide.

[0005] One approach to thermally stabilizing vaccines is to add stabilizing additives to the formulation. Many formulations require complex mixtures of multiple additives, which can add to the cost and complexity of manufacture. The development of a liquid or lyophilized formulation that is stable for extended periods with or without refrigeration would lower manufacturing, distribution, and storage costs, thereby contributing to wider use of vaccines and improved vaccine effectiveness.SUMMARY

[0006] Provided herein, inter alia, are the following embodiments:

[0007] Disclosed herein are liquid, lyophilized, or frozen formulations comprising an effective amount of live-attenuated and / or vector-based virus; about 30 to about 45% (w / v) trehalose; one or more monovalent salts; and a buffer and / or antioxidant comprising histidine.

[0008] Disclosed herein are liquid, lyophilized, or frozen formulations comprising an effective amount of live-attenuated and / or vector-based virus and from about 30% to about 40% (w / v) trehalose, about 100 mM monosodium glutamate (MSG), about 160 mM NaCl, and about 10 mM histidine.

[0009] Disclosed herein are liquid, lyophilized, or frozen formulations comprising an effective amount of live-attenuated respiratory syncytial virus (RSV); about 30 to about 45% (w / v) trehalose; one or more monovalent salts; and a buffer and / or antioxidant comprising histidine.

[0010] Disclosed herein are liquid, lyophilized, or frozen formulations comprising an effective amount of live-attenuated RSV; about 30 to about 45 % (w / v) trehalose; about 10 to about 300 mM NaCl; about 0.5 to about 300 mM monosodium glutamate (MSG) or potassium glutamate; and about 10 to about 100 mM histidine; wherein the formulation has a pH of about 6 to about 8.

[0011] In some embodiments, the virus is enveloped. In some embodiments, the virus is RSV ANS2 / A1313 / 11314L. In some embodiments, the effective amount of the RSVANS2 / A1313 / 11314L is about 5 to about 9 logio plaque forming units (PFU) per dose. In some embodiments, the effective amount of the RSV ANS2 / A1313 / 11314L is about 5 to about 6 logio PFU per dose or about 6.1 to about 7 logio PFU per dose or about 7.1 to about 8 logio PFU per dose or about 8.1 to about 9 logio PFU per dose. In some embodiments, the effective amount of the RSV ANS2 / A1313 / 11314L is about 5.6 logio PFU per dose or about 6.2 logio PFU per dose.

[0012] In some embodiments, a codon in the live-attenuated RSV that encodes a serine at position 1313 of the L protein is deleted resulting in the deletion of the amino acid in the L protein (A I 3 I 3). In some embodiments, an amino acid residue substitution of leucine for isoleucine at position 1314 with reference to SEQ ID NO: 3 in the RSV results in a genetically stabilizing mutation in the L gene (11314L). In some embodiments, the RSV comprises a large polymerase protein (L), a phosphoprotein (P), a nucleocapsid protein (N), an M2-1 protein nonstructural protein 1 (NS1), a glycoprotein (G), a fusion protein (F), a matrix protein (M), an M2-2 protein, and a small hydrophobic protein (SH); and a genome or antigenome comprising a deletion of the codon that encodes the serine at position 1313, or a corresponding position, of the L protein; a mutation of amino acid sequence residue 1314, or a corresponding position, of the L protein, wherein the mutation of L protein amino acid sequence residue 1314 is an amino acid substitution of leucine for isoleucine, wherein the leucine is encoded by a codon set forth as CTG; a deletion of the NS2 gene; and a nucleotide modification at position 14456 with reference to SEQ ID NO: 1 that represents a change from a thymine (T) to an adenine (A).

[0013] In some embodiments, the formulation comprises a liquid formulation. In some embodiments, the formulation comprises a lyophilized formulation. In some embodiments, the liquid formulation is formulated for administration to a human subject. In some embodiments, the administration is intranasal. In some embodiments, the formulation does not comprise a polyoxyethylene-polyoxypropylene (PEO-PPO) block copolymer. In some embodiments, the formulation is a liquid formulation, wherein the liquid formulation is not subsequently dried and reconstituted.

[0014] In some embodiments, the formulation further comprises monosodium glutamate (MSG) or potassium glutamate (PG). In some embodiments, the formulation comprises about 10 to about 100 mM histidine. In some embodiments, the formulation comprises about 30% trehalose. In some embodiments, the formulation comprises about 40% trehalose. In some embodiments, the formulation comprises about 0.5 to about 300 mM monosodium glutamate(MSG) or potassium glutamate (PG). In some embodiments, the monovalent salt is NaCl. In some embodiments, the formulation comprises about 10 to about 300 mM NaCl.

[0015] In some embodiments, the pH is about 6 to about 8. In some embodiments, the pH is about 7 ± 0.5.

[0016] In some embodiments, the live-attenuated virus comprises a paramyxovirus. In some embodiments, the live-attenuated virus comprises a respiratory syncytial virus (RSV), wherein the RSV is for administration to pediatric subjects. In some embodiments, the formulation comprises a vector-based PIV virus, optionally wherein the PIV is PIV3. In some embodiments, the PIV comprises one or more heterologous RSV or hMPV antigens. In some embodiments, the RSV antigen is RSV F and / or RSV G.

[0017] In some embodiments, the formulation is liquid, and wherein there is a titer loss of less than 1 logio PFU / mL when stored at about 2 to about 8 degrees Celsius for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months. In some embodiments, there is a titer loss of less than 1 logio PFU / mL after about 12 to about 24 months in storage at about 2 to about 8 degrees Celsius. In some embodiments, the formulation is liquid, and there is a titer loss of less than 1 logio PFU / mL when stored at about 37 degrees Celsius for at least 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days. In some embodiments, there is a titer loss of less than 1 logio PFU / mL for about 1 to about 2 weeks at 37 °C. In some embodiments, the formulation is liquid, and there is a titer loss of less than 1 logio PFU / mL when stored at room temperature for at least 2-6, 2-5, 2-4, 2- 3, 3-6, 3-5, 3-4, 4-6, 4-5, or 5-6 months. In some embodiments, there is a titer loss of less than1 logio PFU / mL at room temperature for 3-4 months. In some embodiments, the formulation is lyophilized, and wherein there is a titer loss of less than 1 logio PFU / mL for about 1 to about2 weeks at 37 °C. In some embodiments, the formulation is lyophilized, and wherein there is a titer loss of less than 1 logio PFU / mL when stored at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In some embodiments, the infectious titer is not substantially lost upon shear stress, multiple freeze thaws, or reconstitution.

[0018] In some embodiments, the vaccine is formulated to be administered intramuscularly or subcutaneously. In some embodiments, the formulation additionally comprises an adjuvant. In some embodiments, the formulation additionally comprises rHSA, a bivalent salt, and / or amino acids.

[0019] Disclosed herein are methods of increasing the stability of a liquid or lyophilized vaccine at room temperature or at a temperature of about 2 to about 8 degrees Celsius comprising formulating a live-attenuated enveloped virus and / or vector-based virus in a liquidor lyophilized formulation comprising about 30 to about 45% (w / v) trehalose, one or more monovalent salts, and a buffer and / or antioxidant comprising histidine.

[0020] In some embodiments, the concentration of trehalose is about 30% (w / v). In some embodiments, the concentration of trehalose is about 40% (w / v).

[0021] Disclosed herein are methods of immunizing a subject against a viral infection comprising administering the formulation described herein.

[0022] Disclosed herein are liquid, lyophilized, or frozen formulations comprising an effective amount of live-attenuated RSV comprising RSV ANS2 / A1313 / 11314L about 30% to about 40% (w / v) trehalose, about 160 mM NaCl, about 100 mM monosodium glutamate (MSG), and about 10 mM histidine, wherein the formulation has a pH of about 7.

[0023] In some embodiments, the concentration of trehalose is about 30% (w / v). In some embodiments, the concentration of trehalose is about 40% (w / v).

[0024] In some embodiments, the RSV comprises a large polymerase protein (L), a phosphoprotein (P), a nucleocapsid protein (N), an M2-1 protein nonstructural protein 1 (NS1), a glycoprotein (G), a fusion protein (F), a matrix protein (M), an M2-2 protein, and a small hydrophobic protein (SH); and a genome or antigenome comprising a deletion of the codon that encodes the serine at position 1313, or a corresponding position, of the L protein; a mutation of amino acid sequence residue 1314, or a corresponding position, of the L protein, wherein the mutation of L protein amino acid sequence residue 1314 is an amino acid substitution of leucine for isoleucine, wherein the leucine is encoded by a codon set forth as CTG; a deletion of the NS2 gene; and a nucleotide modification at position 14456 with reference to SEQ ID NO: 1 that represents a change from a thymine (T) to an adenine (A).

[0025] The following embodiments are further provided. Embodiment 1 is a liquid, lyophilized, or frozen formulation comprising: a. an effective amount of live-attenuated and / or vector-based virus; b. about 30 to about 45% (w / v) trehalose; c. one or more monovalent salts; and d. a buffer and / or antioxidant comprising histidine.

[0026] Embodiment 2 is a liquid, lyophilized, or frozen formulation comprising an effective amount of live-attenuated and / or vector-based virus and from about 30% to about 40% (w / v) trehalose, about 100 mM monosodium glutamate (MSG), about 160 mM NaCl, and about 10 mM histidine.

[0027] Embodiment 3 is a liquid, lyophilized, or frozen formulation comprising: a. an effective amount of live-attenuated respiratory syncytial virus (RSV);b. about 30 to about 45% (w / v) trehalose; c. one or more monovalent salts; and d. a buffer and / or antioxidant comprising histidine.

[0028] Embodiment 4 is a liquid, lyophilized, or frozen formulation comprising: a. an effective amount of live-attenuated RSV; b. about 30 to about 45 % (w / v) trehalose; c. about 10 to about 300 mM NaCl; d. about 0.5 to about 300 mM monosodium glutamate (MSG) or potassium glutamate; and e. about 10 to about 100 mM histidine; wherein the formulation has a pH of about 6 to about 8.

[0029] Embodiment 5 is the formulation of embodiment 1 or embodiment 2, wherein the virus is enveloped.

[0030] Embodiment 6 is the formulation of any one of embodiments 1-4, wherein the virus is RSV ANS2 / A1313 / 11314L.

[0031] Embodiment 7 is the formulation of embodiment 6, wherein the effective amount of the RSV ANS2 / A1313 / 11314L is about 5 to about 9 logio plaque forming units (PFU) per dose.

[0032] Embodiment 8 is the formulation of embodiment 6, wherein the effective amount of the RSV ANS2 / A1313 / 11314L is about 5 to about 6 logio PFU per dose or about 6.1 to about 7 logio PFU per dose or about 7.1 to about 8 logio PFU per dose or about 8.1 to about 9 logio PFU per dose.

[0033] Embodiment 9 is the formulation of embodiment 6, wherein the effective amount of the RSV ANS2 / A1313 / 11314L is about 5.6 logio PFU per dose or about 6.2 logio PFU per dose.

[0034] Embodiment 10 is the formulation of any one of embodiments 3 or 4, wherein a codon in the live-attenuated RSV that encodes a serine at position 1313 of the L protein is deleted resulting in the deletion of the amino acid in the L protein (A l 3 13).

[0035] Embodiment 11 is the formulation of any one of embodiments 3, 4, or 10, wherein an amino acid residue substitution of leucine for isoleucine at position 1314 with reference to SEQ ID NO: 3 in the RSV results in a genetically stabilizing mutation in the L gene (11314L).

[0036] Embodiment 12 is the formulation of any one of the preceding embodiments, wherein the RSV comprises:a large polymerase protein (L), a phosphoprotein (P), a nucleocapsid protein (N), an M2-1 protein nonstructural protein 1 (NS1), a glycoprotein (G), a fusion protein (F), a matrix protein (M), an M2-2 protein, and a small hydrophobic protein (SH); and a genome or antigenome comprising a deletion of the codon that encodes the serine at position 1313, or a corresponding position, of the L protein; a mutation of amino acid sequence residue 1314, or a corresponding position, of the L protein, wherein the mutation of L protein amino acid sequence residue 1314 is an amino acid substitution of leucine for isoleucine, wherein the leucine is encoded by a codon set forth as CTG; a deletion of the NS2 gene; and a nucleotide modification at position 14456 with reference to SEQ ID NO: 1 that represents a change from a thymine (T) to an adenine (A).

[0037] Embodiment 13 is the formulation of any one of the preceding embodiments, comprising a liquid formulation.

[0038] Embodiment 14 is the formulation of any one of the preceding embodiments, comprising a lyophilized formulation.

[0039] Embodiment 15 is the formulation of embodiment 13, wherein the liquid formulation is formulated for administration to a human subject.

[0040] Embodiment 16 is the formulation of embodiment 15, wherein the administration is intranasal.

[0041] Embodiment 17 is the formulation of any one of the preceding embodiments, wherein the formulation does not comprise a polyoxyethylene-polyoxypropylene (PEO-PPO) block copolymer.

[0042] Embodiment 18 is the formulation of any one of the preceding embodiments, which is a liquid formulation, wherein the liquid formulation is not subsequently dried and reconstituted.

[0043] Embodiment 19 is the formulation of any one of the preceding embodiments, which further comprises monosodium glutamate (MSG) or potassium glutamate (PG).

[0044] Embodiment 20 is the formulation of any one of the preceding embodiments, comprising about 10 to about 100 mM histidine.

[0045] Embodiment 21 is the formulation of any one of the preceding embodiments, which comprises about 30% trehalose.

[0046] Embodiment 22 is the formulation of any one of the preceding embodiments, which comprises about 40% trehalose.

[0047] Embodiment 23 is the formulation of any one of the preceding embodiments, which comprises about 0.5 to about 300 mM monosodium glutamate (MSG) or potassium glutamate (PG).

[0048] Embodiment 24 is the formulation of any one of the preceding embodiments, wherein the monovalent salt is NaCl.

[0049] Embodiment 25 is the formulation of any one of the preceding embodiments, which comprises about 10 to about 300 mM NaCl.

[0050] Embodiment 26 is the formulation of any one of the preceding embodiments, wherein the pH is about 6 to about 8.

[0051] Embodiment 27 is the formulation of any one of the preceding embodiments, wherein the pH is about 7 ± 0.5.

[0052] Embodiment 28 is the formulation of any one of preceding embodiments, wherein the live-attenuated virus comprises a paramyxovirus.

[0053] Embodiment 29 is the formulation of any one of preceding embodiments, wherein the live-attenuated virus comprises a respiratory syncytial virus (RSV).

[0054] Embodiment 30 is the formulation of any one of the preceding embodiments, wherein the virus is RSV, and wherein the RSV is for administration to pediatric subjects.

[0055] Embodiment 31 is the formulation of any one of the preceding embodiments, comprising a vector-based PIV virus, optionally wherein the PIV is PIV3.

[0056] Embodiment 32 is the formulation of embodiment 31, wherein the PIV comprises one or more heterologous RSV or hMPV antigens.

[0057] Embodiment 33 is the formulation of embodiment 32, wherein the RSV antigen is RSV F and / or RSV G.

[0058] Embodiment 34 is the formulation of any one of the preceding embodiments, wherein the formulation is liquid, and wherein there is a titer loss of less than 1 logio PFU / mL when stored at about 2 to about 8 degrees Celsius for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months.

[0059] Embodiment 35 is the formulation of any of the preceding embodiments, wherein there is a titer loss of less than 1 logio PFU / mL after about 12 to about 24 months in storage at about 2 to about 8 degrees Celsius.

[0060] Embodiment 36 is the formulation of any one of the preceding embodiments, wherein the formulation is liquid, and wherein there is a titer loss of less than 1 logio PFU / mL when stored at about 37 degrees Celsius for at least 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days.

[0061] Embodiment 37 is the formulation of any one of the preceding embodiments, wherein there is a titer loss of less than 1 logio PFU / mL for about 1 to about 2 weeks at 37 °C.

[0062] Embodiment 38 is the formulation of any one of the preceding embodiments, wherein the formulation is liquid, and wherein there is a titer loss of less than 1 logio PFU / mL when stored at room temperature for at least 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, or 5-6 months.

[0063] Embodiment 39 is the formulation of any one of the preceding embodiments, wherein there is a titer loss of less than 1 logio PFU / mL at room temperature for 3-4 months.

[0064] Embodiment 40 is the formulation of any one of embodiments 1-33, wherein the formulation is lyophilized, and wherein there is a titer loss of less than 1 logio PFU / mL for about 1 to about 2 weeks at 37 °C.

[0065] Embodiment 41 is the formulation of any one of embodiments 1-33, wherein the formulation is lyophilized, and wherein there is a titer loss of less than 1 logio PFU / mL when stored at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.

[0066] Embodiment 42 is the formulation of any one of the preceding embodiments, wherein the infectious titer is not substantially lost upon shear stress, multiple freeze thaws, or reconstitution.

[0067] Embodiment 43 is the formulation of any one of the preceding embodiments, wherein the vaccine is formulated to be administered intramuscularly or subcutaneously.

[0068] Embodiment 44 is a formulation as embodimented in any one of the preceding embodiments, which additionally comprises an adjuvant.

[0069] Embodiment 45 is a formulation as embodimented in any one of the preceding embodiments, which additionally comprises rHSA, a bivalent salt, and / or amino acids.

[0070] Embodiment 46 is a method of increasing the stability of a liquid or lyophilized vaccine at room temperature or at a temperature of about 2 to about 8 degrees Celsius comprising formulating a live-attenuated enveloped virus and / or vector-based virus in a liquid or lyophilized formulation comprising about 30 to about 45% (w / v) trehalose, one or more monovalent salts, and a buffer and / or antioxidant comprising histidine.

[0071] Embodiment 47 is the method of embodiment 46, wherein the concentration of trehalose is about 30% (w / v).

[0072] Embodiment 48 is the method of embodiment 46, wherein the concentration of trehalose is about 40% (w / v).

[0073] Embodiment 49 is a method of immunizing a subject against a viral infection comprising administering the formulation of any one of embodiments 1-48.

[0074] Embodiment 50 is a liquid, lyophilized, or frozen formulation comprising: a. an effective amount of live-attenuated RSV comprising RSV ANS2 / A1313 / 11314L; b. about 30 to about 40 % (w / v) trehalose; c. about 160 mM NaCl; d. about 100 mM monosodium glutamate (MSG); and e. about 10 mM histidine; wherein the formulation has a pH of about 7.

[0075] Embodiment 51 is the formulation of embodiment 50, wherein the formulation comprises about 30 % (w / v) trehalose.

[0076] Embodiment 52 is the formulation of embodiment 50, wherein the formulation comprises about 40 % (w / v) trehalose.

[0077] Embodiment 53 is the formulation of any one of embodiments 50-52, wherein the live-attenuated RSV comprises: a large polymerase protein (L), a phosphoprotein (P), a nucleocapsid protein (N), an M2-1 protein nonstructural protein 1 (NS1), a glycoprotein (G), a fusion protein (F), a matrix protein (M), an M2-2 protein, and a small hydrophobic protein (SH); and a genome or antigenome comprising a deletion of the codon that encodes the serine at position 1313, or a corresponding position, of the L protein; a mutation of amino acid sequence residue 1314, or a corresponding position, of the L protein, wherein the mutation of L protein amino acid sequence residue 1314 is an amino acid substitution of leucine for isoleucine, wherein the leucine is encoded by a codon set forth as CTG; a deletion of the NS2 gene; and a nucleotide modification at position 14456 with reference to SEQ ID NO: 1 that represents a change from a thymine (T) to an adenine (A).BRIEF DESCRIPTION OF THE DRAWINGS

[0078] FIGs. 1A-C show the experimental design of a formulation development study (FIG. 1A); thermal stability in titer for 1 week at 37 °C of liquid (black bars) and lyophilized (lyo) (white bars) formulations with different trehalose (FIG. IB); the thermal stability in titer loss for 1 week at 37 °C of liquid and lyo formulations with the same trehalose concentration (30%) and different salt concentrations (FIG. 1C). In FIG. 1A, trehalose (10% = -1, 20% = 0, and 30% = 1), NaCl (0 mM = -1, 80 mM = 0, and 160 mM = 1), monosodium glutamate (MSG) (0 mM = -1, 50 mM = 0, and 100 mM = 1), histidine (3.33 mM = -1, 10 mM = 0, and 16.67mM = 1). In FIG. IB, the first bar in each column is “liquid 1 week titer at 37 °C” and the second bar in each column is “lyo 1 week titer at 37 °C”.

[0079] FIGs. 2A and 2B show thermal stability profiles of different % trehalose liquid formulations at 37 °C (FIG. 2A) and 5 °C (FIG. 2B).

[0080] FIGs. 3A and 3B show the real time stability at 5 °C ±3 °C of 30% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0 liquid formulation. FIG. 3 A is high dose drug product toxicology (Tox) lot L5 and FIG. 3B is GMP clinical high dose drug product lot L6.

[0081] FIGs. 4A-4C show a stability study on anRSV ANS2 / A1313 / 11314L drug substance (DS) lot L5 in 30% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0 formulation from a 120 L Phase I manufacturing process. FIG. 4A shows 5 times freeze-thaw results; FIG. 4B is the thawed DS stability at 5 °C ±3 °C, and FIG. 4C is the thawed DS stability at 25 °C ±2 °C.

[0082] FIGs. 5A and 5B show a real-time 36-month stability study at 5 °C ±3 °C comparison between 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0 made from different DS lots. FIG. 5A is high dose drug product stability profile in 40% trehalose liquid formulation, and FIG. 5B is low dose drug product stability profile in 40% trehalose liquid formulation.

[0083] FIGs. 6A-6D show a stability study on an RSV ANS2 / A1313 / 11314L drug substance (DS) lot L8 in 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0 formulation from a 200 L Phase III manufacturing process. FIG. 6A shows 5 times freeze-thaw results; FIG. 6B is the thawed DS stability at 5 °C ±3 °C for up to 90 days, FIG. 6C is the thawed DS stability at 5 °C ±3 °C for up to 130 weeks, and FIG. 6D is the thawed DS stability at 25 °C ±2 °C.

[0084] FIGs. 7A-7G show stability profiles by infectious titer for 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0 liquid drug product. FIG. 7A shows infectious titer for 5 ±3 °C for up to 24 weeks. FIG. 7B shows infectious titer for 5 ±3 °C for up to 24 months. FIG. 7C shows infectious titer for 25 ±2 °C. FIG. 7D shows infectious titer for 37 ±2 °C. FIG. 7E shows infectious titer for 45 ±2 °C. FIG. 7F is formulated bulk product holding stability study by infectious titer at 5 °C for up to 3 months. FIG. 7G is Advanced Kinetics and Technology Solutions (AKTS) modeling and shelf-life predictions based on 3- month data. The best fit model selected is Model #53 competitive 2-steps kinetics, best widely applicable information criterion (wAIC), known as Watanabe- Akaike information criterion, is the generalized version of the Akaike information criterion (AIC) onto singular statisticalmodels and widely applicable Bayesian information criterion (wBIC) is the generalized version of Bayesian information criterion (BIC) onto singular statistical models.

[0085] FIGs. 8A-8D show 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0 liquid formulation stability comparison between drug product made from DS Lot L7 and DS Lot L9 at 5 °C (FIG. 8B, up to 240 days and FIG. 8C, up to 24 months) and at 37°C (FIG. 8A). FIG. 8D is the AKTS shelf-life prediction comparison in titer loss at 5 °C.

[0086] FIGs. 9A-C show thermal stability of addition of recombinant human serum albumin (rHSA) study. FIG. 9 A is DS stability comparison without and with rHSA. FIG. 9B is DP stability comparison without and with low concentration rHSA at 0.5 mg / mL. FIG. 9C is DP stability comparison without and with high concentration rHSA at 1.4 mg / mL.

[0087] FIGs. 10A-10E show the candidate formulations stability profiles including the accelerated stability testing. FIG. 10A shows infectious titer for 5 ±3 °C. FIG. 10B shows infectious titer for 25 ±2 °C. FIG. 10C shows infectious titer for 37 ±2 °C. FIG. 10D is the AKTS shelf-life prediction of low concentration rHSA formulation in titer loss at 5 °C. The best fit model selected is Model #43 1-step kinetics, best wAIC and wBIC. FIG. 10E is the AKTS shelf-life prediction of high concentration rHSA formulation in titer loss at 5 °C. The best fit model selected is Model #34 1-step kinetics, best wAIC and wBIC.

[0088] FIG. 11 is thermal study profile comparison of 40% trehalose without and with new excipient hydroxyethyl starch (HES).

[0089] FIGs. 12A-12F show the candidate formulations with HES stability profiles including the accelerated stability testing. FIG. 12A shows infectious titer for 5 ±3 °C at up to 24 weeks (6 months). FIG. 12B shows infectious titer for 5 ±3 °C at up to 104 weeks (24 months). FIG. 12C shows infectious titer for 25 ±2 °C. FIG. 12D shows infectious titer for 37 ±2 °C. FIG. 12E shows infectious titer for 45 ±2 °C. FIG. 12F is the AKTS shelf-life prediction of 2.5% concentration HES formulation in titer loss at 5 °C based on 6-month data. The best fit model selected is Model #56 2-steps kinetics, best wAIC and wBIC.

[0090] FIG. 13 shows summary of thermal stability comparison from all dried formulations.

[0091] FIG. 14 shows results of an exemplary herpes simplex type 2 (HSV-2) vaccine lyo formulation study.

[0092] FIG. 15 illustrates the genome structure of RSV ANS2 / A1313 / 11314L.DETAILED DESCRIPTION

[0093] The present disclosure provides liquid and dried formulations that have improved yield and stability and methods for preparing them, wherein the vaccine comprises a live- attenuated enveloped virus greater than about 15% (w / v) trehalose, a monovalent salt, and histidine, optionally wherein the virus is RSV. The formulations and methods provide at least the advantages of improving the liquid and dried stability profile, and integration of a liquid vaccine in a pre-filled syringe or device for immediate administration without reconstitution. In some embodiments, the device is for intranasal delivery. In some embodiments, the device is pre-filled with the liquid formulation for intranasal delivery. In some embodiments, the formulation provided herein is liquid and is stable when stored at about 2 to about 8 degrees Celsius for at least 12-24 months with a titer loss of less than 1 logio PFU / mL. In some embodiments, the formulation provided herein is liquid and under thermal stress conditions the formulation can maintain a titer loss of less than 1 logio PFU / mL when stored at about 37 degrees Celsius for at least 12-25 days. In some embodiments, the formulation provided herein is liquid and is stable when stored at room temperature (typically about 25 degrees Celsius) for at least 3-6 months with a titer loss of less than 1 logio PFU / mL.

[0094] The present invention is based, in part, on the surprising stability obtained when formulating live-attenuated viral vaccines with high concentrations of trehalose.I. Definitions

[0095] Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings:

[0096] “Or” is used in the inclusive sense, i.e., equivalent to “and / or,” unless the context requires otherwise.

[0097] As used herein, a “live-attenuated virus” is a virus that is viable and demonstrates reduced, weakened, or no clinical signs of disease when administered to a subject. A “live- attenuated vaccine” comprises a live-attenuated virus.

[0098] A “vector” or “vector-based” virus or vaccine, as used herein, comprises a virus that is modified to express one or more heterologous antigens. For example, vaccinia viruses have been used as vaccine vectors to express influenza genes for use in immunizing against influenza. De Vries and Rimmelzwaan; Human Vaccine Immunother. (2016) Nov; 12(11): 2881-2901. Similarly, parainfluenza viruses (PIVs) have been used as vectors to express non- PIV viral genes, such as, for example, RSV, herpes simplex virus (HSV), and human metapneumovirus (hMPV) for use in immunizing against RSV, HSV, and hMPV, for example.Further, PIVs of one species have been used as vectors to express PIV viral genes of another species. In each instance, such vaccine is considered a “vector” or “vector-based” vaccine and the recombinant virus a “vector” or “vector-based” virus. In some instances, a “vector” or “vector-based” vaccine is also a live-attenuated vaccine where the vaccine demonstrates reduced, weakened, or no clinical signs of disease when administered to a subject.

[0099] “Prevention,” as used herein, refers to prophylaxis, avoidance of disease manifestation, a delay of onset, and / or reduction in frequency and / or severity of one or more symptoms of a particular disease, disorder, or condition (e.g., infection with RSV). In some embodiments, prevention is assessed on a population basis such that an agent is considered to “prevent” a particular disease, disorder, or condition if a statistically significant decrease in the development, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition.

[0100] As used herein, the term “vaccination” or “vaccinate” refers to the administration of a composition intended to generate an immune response, for example, to a disease-causing agent. Vaccination can be administered before, during, and / or after exposure to a diseasecausing agent, and / or to the development of one or more symptoms, and in some embodiments, before, during, and / or shortly after exposure to the agent. In some embodiments, vaccination includes multiple administrations, appropriately spaced in time, of a vaccinating composition.

[0101] As used herein, “RSV ANS2 / A1313 / 11314L” refers to an RSV ANS2 / A1313 / 11314L (NIH) or an RSV ANS2 / A1313 / 11314L (Sanofi). Each of the ANS2 / A1313 / 11314L (NIH) and the RSV ANS2 / A1313 / 11314L (Sanofi) comprise a live- attenuated RSV with (i) a 523 nucleotide (nt) deletion of the NS2 gene (ANS2), (ii) an amino acid deletion in the L protein, and (iii) a genetically stabilizing mutation in the L gene (see FIG. 15). The live-attenuated RSV of the RSV ANS2 / A1313 / 11314L (Sanofi) also includes a nucleotide modification at position 14456 with reference to SEQ ID NO: 1 that represents a change from a thymine (T) to an adenine (A) in a non-coding region.

[0102] As used herein, “RSV ANS2 / A1313 / 11314L vaccine” refers to an “RSV ANS2 / A1313 / 11314L (NIH) vaccine” or an “RSV ANS2 / A1313 / 11314L (Sanofi) vaccine.” An RSV ANS2 / A1313 / 11314L (NIH) vaccine comprises an effective amount of RSV ANS2 / A1313 / 11314L (NIH). An RSV ANS2 / A1313 / 11314L (Sanofi) vaccine comprises an effective amount of RSV ANS2 / A1313 / 11314L (Sanofi).

[0103] “Immune response,” as used herein, refers to a response of a cell of the immune system, such as a B cell, T cell, dendritic cell, macrophage, or polymorphonucleocyte, to a stimulus such as an antigen or vaccine. An immune response can include any cell of the bodyinvolved in a host defense response, including, for example, an epithelial cell that secretes an interferon or a cytokine. An immune response includes, but is not limited to, an innate and / or adaptive immune response. As used herein, a “protective immune response” refers to an immune response that protects a subject from infection (e.g., prevents infection or prevents the development of disease associated with infection). Methods of measuring immune responses include, for example, measuring proliferation and / or activity of lymphocytes (such as B or T cells), secretion of cytokines or chemokines, inflammation, antibody production, and the like. An “antibody response” is an immune response in which antibodies are produced.

[0104] “Immunizing” and the like, as used herein, refer to the act of eliciting an immune response in a subject or protecting a subject against infection.

[0105] “Adjuvant,” as used herein, refers to a substance or vehicle that non-specifically enhances the immune response to an antigen. Adjuvants can include, without limitation, a suspension of minerals (e.g., alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; a water-in-oil or oil-in-water emulsion in which antigen solution is emulsified in mineral oil or in water (e.g., Freund’s incomplete adjuvant). Sometimes, killed mycobacteria is included (e.g., Freund’s complete adjuvant) to further enhance antigenicity. Immunostimulatory oligonucleotides (e.g., a CpG motif) can also be used as adjuvants (for example, see U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants can also include biological molecules, such as Toll-like receptor (TLR) agonists and costimulatory molecules. Exemplary biological adjuvants include, but are not limited to, IL-2, RANTES, GM-CSF, TNF-a, IFN-y, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL, or combinations thereof.

[0106] As used herein, “liquid” is given its traditional meaning and “frozen liquid” is a solid state liquid distinguished from liquid state.

[0107] “Formulation” refers to a composition containing an active pharmaceutical or biological ingredient, along with one or more additional components. The term “formulation” may be used interchangeably with the terms “pharmaceutical composition,” “vaccine composition,” and “vaccine formulation” herein. Additional components that may be included as appropriate include pharmaceutically acceptable excipients, additives, diluents, buffers, sugars, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), chelating agents, surfactants, polyols, bulking agents, stabilizers, lyoprotectants, solubilizers, emulsifiers, salts, adjuvants, tonicity enhancing agents (such as alkali metal halides, e.g., sodium or potassium chloride, mannitol, or sorbitol), delivery vehicles, and anti-microbial preservatives.

[0108] As used herein, being “stable” or “stabilized” with respect to an active ingredient in a formulation means a loss of less than 1 logio PFU / ml at a given temperature and time. Stability can be measured using the assay described herein, as well as assays known in the art that are used to measure activity or potency or virus infectious titer.

[0109] As used herein, “treatment” refers to any administration or application of a therapeutic for disease or disorder in a subject, and includes inhibiting the disease, arresting its development, relieving one or more symptoms of the disease, curing the disease, or preventing reoccurrence of one or more symptoms of the disease.

[0110] As used herein, a “therapeutically effective amount” or “effective amount” is the amount of a composition, or an active component thereof, sufficient to provide a beneficial effect or to otherwise reduce a detrimental nonbeneficial event to the individual to whom the composition is administered. By “therapeutically effective dose” or “effective dose,” as used herein, is meant a dose that produces one or more desired or desirable (e.g., beneficial) effects for which it is administered, such administration occurring one or more times over a given period of time. The exact dose will depend on the purpose of the treatment and will be ascertainable using known techniques.

[0111] The term “approximately” or “about” is used herein to mean roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower). In some embodiments, the term indicates deviation from the indicated numerical value by ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±10%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±5%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±4%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±3%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±2%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±1%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.9%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.8%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.7%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.6%. In some embodiments, “about” indicates deviation from theindicated numerical value by ±0.5%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.4%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.3%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.2%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.1%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.05%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.01%. All ranges set forth herein are intended to be inclusive of the lower and upper limit of the range.

[0112] The term “pediatric subject” is a subject aged 21 or younger at the time of immunization. Pediatric subpopulations are further characterized as: (i) neonates - from birth through the first 28 days of life; (ii) infants and toddlers - from 29 days to less than 2 years; (iii) children - 2 years to less than 12 years; and (iv) adolescents - aged 12 years through 21 years. In some aspects, the pediatric subject may be 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year(s) of age or less. In some aspects, the pediatric subject may be at least 6 months of age. In some aspects, the pediatric subject may be 6 months to 18 years of age. In some aspects, the pediatric subject may be 6 months to 10 years of age. In some aspects, the pediatric subject may be 4 months to 4 years of age. In some aspects, the pediatric subject may be 6 to 18 months of age. In some aspects, the pediatric subject may be from 6 months to less than 24 months of age. Additional age ranges may also be included for pediatric subjects.

[0113] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention is described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims and included embodiments.

[0114] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a conjugate” includes a plurality of conjugates and reference to “a cell” includes a plurality of cells and the like.

[0115] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits andthe error associated with the measurement. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings.

[0116] Unless specifically noted in the specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of’ or “consisting essentially of’ the recited components; embodiments in the specification that recite “consisting of’ various components are also contemplated as “comprising” or “consisting essentially of’ the recited components; and embodiments in the specification that recite “consisting essentially of’ various components are also contemplated as “consisting of’ or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims). The term “or” is used in an inclusive sense, ie., equivalent to “and / or,” unless the context clearly indicates otherwise.

[0117] The section headings used herein are for organizational purposes only and are not to be construed as limiting the desired subject matter in any way. In the event that any material incorporated by reference contradicts any term defined in this specification or any other express content of this specification, this specification controls. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.II. Vaccine Formulations

[0118] A. Formulations

[0119] Formulations described herein may be liquid, lyophilized, or frozen. Liquid formulations may be prepared as a liquid (z.e., never been lyophilized) and be suitable for longterm storage at 2-8 °C over the product shelf-life, may be reconstituted after lyophilization, or thawed after freezing, and stored at room temperature more than hours or days. In some embodiments, the formulation may be stored lyophilized at 2-8 °C or room temperature prior to reconstitution. In some embodiments, the formulation does not comprise a polyoxyethylenepolyoxypropylene (PEO-PPO) block copolymer or other block copolymers considered similar to PEO-PPO by those of skill in the art.

[0120] In some embodiments, the formulation is liquid and the formulation is stable when stored at about 2 to about 8 degrees Celsius (°C) for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months. In some embodiments, being “stable” means a loss of less than 1 logio PFU / ml at a given temperature and time. See, also, Guidelines on the Stability Evaluation of Vaccines, Geneva: World Health Organization; 2006. 28 p. Report No.: WHO / BS / 06.2049 Final; and WHO Technical Report Series No. 962: Guidelines on the Stability Evaluation of Vaccines, Geneva: World Health Organization; 2011. 28. Report No.: 57, incorporated herein by reference, which provides guidelines for determining stable formulations. In some such embodiments, there is a titer loss of less than 1 logio PFU / mL after about 12-24 months in storage at about 2 to about 8 degrees Celsius.

[0121] In some embodiments, the formulation is liquid and under thermal stress conditions the formulation can maintain a titer loss of under 1 logio PFU / mL when stored at about 37 degrees Celsius for at least 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days. In some such embodiments, there is a titer loss of less than 1 logio PFU / mL for 1-2 weeks at 37 degrees Celsius.

[0122] In some embodiments, the formulation is liquid and there is a titer loss of less than 1 logio PFU / mL when stored at room temperature for at least 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, or 5-6 months. In some embodiments, the formulation is liquid and is stable when stored at room temperature for at least 3 months. In some embodiments, the formulation is liquid and is stable when stored at room temperature for at least 4 months. In some embodiments, the formulation is lyophilized, and wherein there is a titer loss of less than 1 logio PFU / mL for about 1 to about 2 weeks at 37 °C. In some embodiments, the formulation is lyophilized, and wherein there is a titer loss of less than 1 logio PFU / mL when stored at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.

[0123] In some embodiments, the infectious titer of the formulation is not substantially lost upon shear stress, multiple freeze thaws, or reconstitution.

[0124] Trehalose

[0125] In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising a live-attenuated enveloped virus and trehalose. In some embodiments, a liquid or lyophilized formulation is provided comprising a live-attenuated enveloped virus and trehalose. In some embodiments, the concentration of trehalose is between about 15% and 60% w / v. In some embodiments, the concentration of trehalose is between about 20% and 55% w / v. In some embodiments, the concentration of trehalose is between about 25% and 50% w / v. In some embodiments, the concentration of trehalose is between about 30% and 45% w / v. In some embodiments, the concentration of trehalose is between about 35% and 40% w / v. In some embodiments, the concentration of trehalose is between about 30% and 40% w / v. In someembodiments, the concentration of trehalose is between about 30% and 35% w / v. In some embodiments, the concentration of trehalose is between about 15% and 20%, 15% and 25%, 15% and 30%, 15% and 35%, 15% and 40%, or 15% and 45% w / v. In some embodiments, the concentration of trehalose is greater than about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45%. In some embodiments, the concentration of trehalose is between about 20% and 25%, 20% and 30%, 20% and 35%, 20% and 40%, or 20% and 45% w / v. In some embodiments, the concentration of trehalose is between about 25% and 30%, 25% and 35%, 25% and 40%, or 25% and 45% w / v. In some embodiments, the concentration of trehalose is between about 30% and 35%, 30% and 40%, or 30% and 45% w / v. In some embodiments, the concentration of trehalose is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45% w / v. In some embodiments, the concentration of trehalose is about 20% w / v. In some embodiments, the concentration of trehalose is about 25% w / v. In some embodiments, the concentration of trehalose is about 30% w / v. In some embodiments, the concentration of trehalose is about 35% w / v. In some embodiments, the concentration of trehalose is about 40% w / v. In some embodiments, the concentration of trehalose is about 45% w / v. In some embodiments, the trehalose may function as a stabilizer. In certain embodiments, the trehalose may function as a lyoprotectant. In various embodiments, the trehalose may function as a stabilizer and a lyoprotectant.

[0126] Salts

[0127] In some embodiments, the formulation further comprises a salt. In some embodiments, the salt is a monovalent salt. In some embodiments, the salt is a bivalent salt. a. Monovalent Salts (e.g., NaCl)

[0128] In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising a live-attenuated enveloped virus, trehalose, and a monovalent salt. In some embodiments, a liquid or lyophilized formulation is provided comprising a live-attenuated enveloped virus, trehalose, and a monovalent salt. In some embodiments, the monovalent salt is sodium chloride (NaCl). In some embodiments, the monovalent salt is monosodium glutamate (MSG) or potassium glutamate (PG; e.g., monopotassium glutamate (MPG)).

[0129] In some embodiments, the concentration of monovalent salt is between about 10 and 300 mM. In some embodiments, the concentration is between about: 10 and 290 mM, 20 and 180 mM, 30 and 170 mM, 40 and 160 mM, 50 and 150 mM, 60 and 140 mM, 70 and 130 mM, 80 and 120 mM, or 90 and 110 mM. In some embodiments, the concentration is between about: 100 and 200 mM, 110 and 200 mM, 120 and 200 mM, 130 and 200 mM, 140and 200 mM, 150 and 200 mM, or 160 and 200 mM. In some embodiments, the concentration is between about 100 and 200 mM, 100 and 190 mM, 100 and 180 mM, 100 and 170 mM, 100 and 160 mM, or 100 and 150 mM. In some embodiments, the concentration is about 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, or 200 mM. In some embodiments, the concentration is about 150 mM. In some embodiments, the concentration is about 160 mM. In some embodiments, the concentration is about 170 mM. In some embodiments, the monovalent salt is NaCl and the concentration of the monovalent salt is about 160 mM.

[0130] In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising: a live-attenuated enveloped virus; about 30-45% w / v trehalose; and about 160 mM monovalent salt, optionally wherein the monovalent salt is NaCl. In some embodiments, a liquid or lyophilized formulation is provided comprising: a live-attenuated enveloped virus; about 30-45% w / v trehalose; and about 160 mM monovalent salt, optionally wherein the monovalent salt is NaCl. In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising: a live-attenuated enveloped virus; about 30% w / v trehalose; and about 160 mM monovalent salt, optionally wherein the monovalent salt is NaCl. In some embodiments, a liquid or lyophilized formulation is provided comprising: a live-attenuated enveloped virus; about 30% w / v trehalose; and about 160 mM monovalent salt, optionally wherein the monovalent salt is NaCl. In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising: a live-attenuated enveloped virus; about 40% w / v trehalose; and about 160 mM monovalent salt, optionally wherein the monovalent salt is NaCl. In some embodiments, a liquid or lyophilized formulation is provided comprising: a live- attenuated enveloped virus; about 40% w / v trehalose; and about 160 mM monovalent salt, optionally wherein the monovalent salt is NaCl. In some embodiments the virus is RSV. In certain embodiments, the vaccine is an RSV ANS2 / A1313 / 11314L vaccine.

[0131] b. Bivalent Salts (e.g.. MgCh or CaCh)

[0132] In some embodiments, the formulation further comprises a bivalent salt, optionally wherein the bivalent salt is MgCh or CaCh. In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising: a live-attenuated enveloped virus; trehalose; a monovalent salt, optionally wherein the monovalent salt is NaCl; a stabilizer, optionally wherein the stabilizer is MSG or PG; histidine; and a bivalent salt, optionally wherein the bivalent salt is MgCh or CaCh. In some embodiments, a liquid or lyophilized formulation is provided comprising: a live-attenuated enveloped virus; trehalose; a monovalent salt, optionally wherein the monovalent salt is NaCl; a stabilizer, optionally wherein the stabilizeris MSG or PG; histidine; and a bivalent salt, optionally wherein the bivalent salt is MgCh or CaCh.

[0133] In some embodiments, the concentration of the bivalent salt (e.g., MgCh or CaCh) is between about 0 and 50 mM. In some embodiments, the concentration of the bivalent salt (e.g., MgCh or CaCh) is between about: 0 and 10 mM, 0 and 20 mM, 0 and 30 mM, 0 and 40 mM, or 0 and 50 mM. In some embodiments, the concentration of the bivalent salt (e.g., MgCh or CaCh) is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or 55 mM. In some embodiments, the concentration of the bivalent salt (e.g., MgCh or CaCh) is about 10 mM.

[0134] In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising: a live-attenuated enveloped virus; about 30-45% w / v trehalose; about 160 mM monovalent salt, optionally wherein the monovalent salt is NaCl; about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG; about 10 mM histidine; and about 0-50 mM bivalent salt, optionally wherein the bivalent salt is MgCh or CaCh. In some embodiments, a liquid or lyophilized formulation is provided comprising: a live-attenuated enveloped virus; about 30-45% w / v trehalose; about 160 mM monovalent salt, optionally wherein the monovalent salt is NaCl; about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG; about 10 mM histidine; and about 0-50 mM bivalent salt, optionally wherein the bivalent salt is MgCh or CaCh. In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising: a live-attenuated enveloped virus; about 30% w / v trehalose; about 160 mM monovalent salt, optionally wherein the monovalent salt is NaCl; about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG; about 10 mM histidine; and about 0-50 mM bivalent salt, optionally wherein the bivalent salt is MgCh or CaCh. In some embodiments, a liquid or lyophilized formulation is provided comprising: a live-attenuated enveloped virus; about 30% w / v trehalose; about 160 mM monovalent salt, optionally wherein the monovalent salt is NaCl; about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG; about 10 mM histidine; and about 0-50 mM bivalent salt, optionally wherein the bivalent salt is MgCh or CaCh. In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising: a live-attenuated enveloped virus; about 40% w / v trehalose; about 160 mM monovalent salt, optionally wherein the monovalent salt is NaCl; about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG; about 10 mM histidine; and about 0-50 mM bivalent salt, optionally wherein the bivalent salt is MgCh or CaCh. In some embodiments, a liquid or lyophilized formulation is provided comprising: a live-attenuated enveloped virus; about 40% w / v trehalose; about 160 mM monovalent salt, optionally wherein the monovalentsalt is NaCl; about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG; about 10 mM histidine; and about 0-50 mM bivalent salt, optionally wherein the bivalent salt is MgCh or CaCh. In some embodiments the virus is RSV. In some embodiments, the vaccine is an RSV ANS2 / A1313 / 11314L vaccine.

[0135] Stabilizers

[0136] In some embodiments, the formulation further comprises a stabilizer. In some embodiments, the stabilizer is monosodium glutamate (MSG) or potassium glutamate (PG). In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising: a live- attenuated enveloped virus; trehalose; a salt, optionally wherein the salt is NaCl; and a stabilizer, optionally wherein the stabilizer is MSG or PG. In some embodiments, a liquid or lyophilized formulation is provided comprising: a live-attenuated enveloped virus; trehalose; a salt, optionally wherein the salt is NaCl; and a stabilizer, optionally wherein the stabilizer is MSG or PG.

[0137] In some embodiments, the concentration of stabilizer is between about 0.5 and 300 mM. In some embodiments, the concentration of stabilizer is between about: 10 and 290 mM, 20 and 180 mM, 30 and 170 mM, 40 and 160 mM, 50 and 150 mM, 60 and 140 mM, 70 and 130 mM, 80 and 120 mM, or 90 and 110 mM. In some embodiments, the concentration of stabilizer is between about: 100 and 200 mM, 110 and 200 mM, 120 and 200 mM, 130 and 200 mM, 140 and 200 mM, 150 and 200 mM, or 160 and 200 mM. In some embodiments, the concentration of stabilizer is between about 100 and 200 mM, 100 and 190 mM, 100 and 180 mM, 100 and 180 mM, 100 and 170 mM, 100 and 160 mM, or 100 and 150 mM. In some embodiments, the concentration of stabilizer is between about 50 and 100 mM. In some embodiments, the concentration of stabilizer is about 80, 85, 90, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 mM. In some embodiments, the concentration of stabilizer is about 100 mM.

[0138] In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising: a live-attenuated enveloped virus; about 30-45% w / v trehalose; about 160 mM salt, optionally wherein the salt is NaCl; and about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG. In some embodiments, a liquid or lyophilized formulation is provided comprising: a live-attenuated enveloped virus; about 30-45% w / v trehalose; about 160 mM salt, optionally wherein the salt is NaCl; and about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG. In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising: a live-attenuated enveloped virus; about 30% w / v trehalose; about 160 mM salt, optionally wherein the salt is NaCl; and about 100 mM stabilizer, optionally whereinthe stabilizer is MSG or PG. In some embodiments, a liquid or lyophilized formulation is provided comprising: a live-attenuated enveloped virus; about 30% w / v trehalose; about 160 mM salt, optionally wherein the salt is NaCl; and about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG. In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising: a live-attenuated enveloped virus; about 40% w / v trehalose; about 160 mM salt, optionally wherein the salt is NaCl; and about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG. In some embodiments, a liquid or lyophilized formulation is provided comprising: a live-attenuated enveloped virus; about 40% w / v trehalose; about 160 mM salt, optionally wherein the salt is NaCl; and about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG. In some embodiments the virus is RSV. In some embodiments, the vaccine is an RSV ANS2 / A1313 / 11314L vaccine.

[0139] Buffer and / or Antioxidant Histidine

[0140] In some embodiments, the formulation further comprises a buffer and / or antioxidant comprising histidine. In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising: a live-attenuated enveloped virus; trehalose; a salt, optionally wherein the salt is NaCl; a stabilizer, optionally wherein the stabilizer is MSG or PG; and a buffer and / or antioxidant comprising histidine. In some embodiments, a liquid or lyophilized formulation is provided comprising: a live-attenuated enveloped virus; trehalose; a salt, optionally wherein the salt is NaCl; a stabilizer, optionally wherein the stabilizer is MSG or PG; and a buffer and / or antioxidant comprising histidine. In some embodiments, histidine has a buffer function. In certain embodiments, histidine has an antioxident function. In various embodiments, histidine has buffer and antioxidant functions. Stated another way, histidine may have a buffer and antioxidant dual function.

[0141] In some embodiments, the concentration of histidine is between about 10 and 100 mM. In some embodiments, the concentration of histidine is between about: 10 and 90 mM, 20 and 80 mM, 30 and 70 mM, or 40 and 60 mM. In some embodiments, the concentration of histidine is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 mM. In some embodiments, the concentration of histidine is about 10 mM.

[0142] In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising: a live-attenuated enveloped virus; about 30-45% w / v trehalose; about 160 mM salt, optionally wherein the salt is NaCl; about 100 mM stabilizer, optionally wherein the histidine is MSG or PG; and about 10 mM histidine. In some embodiments, a liquid or lyophilized formulation is provided comprising: a live-attenuated enveloped virus; about 30-45% w / v trehalose; about 160 mM salt, optionally wherein the salt is NaCl; about 100 mM stabilizer, optionally wherein the histidine is MSG or PG; and about 10 mM histidine. In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising: a live- attenuated enveloped virus; about 30% w / v trehalose; about 160 mM salt, optionally wherein the salt is NaCl; about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG; and about 10 mM histidine. In some embodiments, a liquid or lyophilized formulation is provided comprising: a live-attenuated enveloped virus; about 30% w / v trehalose; about 160 mM salt, optionally wherein the salt is NaCl; about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG; and about 10 mM histidine. In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising: a live-attenuated enveloped virus; about 40% w / v trehalose; about 160 mM salt, optionally wherein the salt is NaCl; about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG; and about 10 mM histidine. In some embodiments, a liquid or lyophilized formulation is provided comprising: a live-attenuated enveloped virus; about 40% w / v trehalose; about 160 mM salt, optionally wherein the salt is NaCl; about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG; and about 10 mM histidine. In some embodiments the virus is RSV. In some embodiments, the vaccine is an RSV ANS2 / A1313 / 11314L vaccine.

[0143] Amino Acids

[0144] In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising a live-attenuated enveloped virus, trehalose, and optionally one or more amino acids. In some embodiments, a liquid or lyophilized formulation is provided comprising a live- attenuated enveloped virus, trehalose, and optionally one or more amino acids.

[0145] The amino acid can be alanine, arginine, aspartic acid, asparagine, cysteine, leucine, isoleucine, methionine, proline, serine, lysine, histidine, glycine, glutamic acid, phenylalanine, threonine, tryptophan, tyrosine, valine, and combinations thereof. In certain embodiments, the amino acid, or combination thereof, is present at a concentration ranging from about 0.1% to about 10% (w / v). Amino acids can also be provided by enzymatic digests of proteins. For example, N-Z-Amine™ A, an enzymatic digest of casein, can be used to provide a combination of amino acids. In some embodiments the virus is RSV. In some embodiments, the vaccine is an RSV ANS2 / A1313 / 11314L vaccine.

[0146] rHSA

[0147] In some embodiments, the formulation further comprises albumin, optionally wherein the albumin is human serum albumin (HSA), optionally, wherein the human serum albumin is recombinant human serum albumin (rHSA). In some embodiments, a liquid,lyophilized, or frozen formulation is provided comprising: a live-attenuated enveloped virus; trehalose; a salt, optionally wherein the salt is NaCl; a stabilizer, optionally wherein the stabilizer is MSG or PG; histidine, optionally MgCh or CaCh; and optionally HSA. In some embodiments, a liquid or lyophilized formulation is provided comprising: a live-attenuated enveloped virus; trehalose; a salt, optionally wherein the salt is NaCl; a stabilizer, optionally wherein the stabilizer is MSG or PG; histidine, optionally MgCh or CaCh; and optionally HSA.

[0148] In some embodiments, the concentration of HSA is between about 0 and 20 mg / mL. In some embodiments, the concentration of HSA is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / mL. In some embodiments, the concentration of HSA is about 5, 10, 15, or 20 mg / mL.

[0149] In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising: a live-attenuated enveloped virus; about 30-40% w / v trehalose; about 160 mM salt, optionally wherein the salt is NaCl; about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG; about 10 mM histidine; about 0-50 mM MgCh or CaCh; and about 0-20 mg / mL rHSA. In some embodiments, a liquid or lyophilized formulation is provided comprising: a live-attenuated enveloped virus; about 30-40% w / v trehalose; about 160 mM salt, optionally wherein the salt is NaCl; about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG; about 10 mM histidine; about 0-50 mM MgCh or CaCh; and about 0-20 mg / mL rHSA. In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising: a live-attenuated enveloped virus; about 30% w / v trehalose; about 160 mM salt, optionally wherein the salt is NaCl; about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG; about 10 mM histidine; about 0-50 mM MgCh or CaCh; and about 0-20 mg / mL rHSA. In some embodiments, a liquid or lyophilized formulation is provided comprising: a live-attenuated enveloped virus; about 30% w / v trehalose; about 160 mM salt, optionally wherein the salt is NaCl; about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG; about 10 mM histidine; about 0-50 mM MgCh or CaCh; and about 0-20 mg / mL rHSA. In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising: a live-attenuated enveloped virus; about 40% w / v trehalose; about 160 mM salt, optionally wherein the salt is NaCl; about 100 mM stabilizer, optionally wherein the stabilizer is MSG or PG; about 10 mM histidine; about 0-50 mM MgCh or CaCh; and about 0-20 mg / mL rHSA. In some embodiments, a liquid or lyophilized formulation is provided comprising: a live-attenuated enveloped virus; about 40% w / v trehalose; about 160 mM salt, optionally wherein the salt is NaCl; about 100 mM stabilizer, optionally wherein the stabilizer is MSG orPG; about 10 mM histidine; about 0-50 mM MgCh or CaCh; and about 0-20 mg / mL rHSA. In some embodiments the virus is RSV. In some embodiments, the vaccine is an RSV ANS2 / A1313 / 11314L vaccine.

[0150] B. Viruses

[0151] Any live-attenuated vaccine may be formulated as provided herein. Also encompassed are vector-based vaccines, which in some instances may also be considered live- attenuated vaccines. In some instances, live-attenuated vaccines use a weakened strain of the virus that causes the disease. In some instances, live-attenuated vaccines are considered attenuated because they are administered to a host species that does not mount an appreciable immune response to the virus despite the virus being able to mount an immune response in another host species. Each of the viruses discussed herein may be present in a live-attenuated formulation or in a vector-based formulation. In embodiments comprising a vector-based formulation, the described virus may be the carrier virus comprising a heterologous viral antigen or may be the source of the heterologous viral antigen.

[0152] In some embodiments, the virus is an enveloped virus. In some embodiments, the virus is a live-attenuated enveloped virus. In some embodiments, the virus is an RNA virus. In some embodiments, the virus is a DNA virus. In some embodiments, the virus is an enveloped DNA virus. In some embodiments, the virus is an enveloped RNA virus.

[0153] In some embodiments, the virus is of the Paramyxoviridae family, which is a family of negative-sense, enveloped, single stranded RNA viruses having genomes of about 13-19 kb. In some embodiments, the paramyxovirus is a respiratory syncytial virus (RSV). In some embodiments, the RSV is human RSV (also known as Human orthopneumovirus).

[0154] In some embodiments, the virus is RSV and comprises a formulation for administration to pediatric subjects (RSV ANS2 / A1313 / 11314L) as disclosed, for example, in WO 2017100756 Al, which is incorporated by reference in its entirety. In some embodiments, the virus is RSV and comprises an attenuated RSV with point mutations, as described, for example, in WO 2013154728 Al, which is incorporated by reference in its entirety. In some embodiments, the virus is RSV and comprises mutations in the RSV open reading frames that interfere with RSV protein expression, as described, for example, in WO 2017100759 Al, which is incorporated by reference in its entirety. In some vector-based formulation embodiments, the heterologous antigen is derived from an RSV virus, optionally wherein the antigen is RSV F and / or RSV G. In some embodiments, the virus is RSV and comprises a formulation for administration to pediatric subjects (RSV ANS2 / A1313 / 11314L) that is a recombinant infectious respiratory syncytial virus including a large polymerase protein (L),phosphoprotein (P), nucleocapsid protein (N), an M2-1 protein nonstructural protein 1 (NS1), a glycoprotein (G), a fusion protein (F), a matrix protein (M), an M2-2 protein, and a small hydrophobic protein (SH), and a genome or antigenome having: a deletion of the codon that encodes the serine at position 1313, or a corresponding position, of the L protein, a mutation of amino acid sequence residue 1314, or a corresponding position, of the L protein, wherein the mutation of L protein amino acid sequence residue 1314 is an amino acid substitution of leucine for isoleucine, wherein the leucine is encoded by a codon set forth as CTG and a deletion of the NS2 gene, as described, for example, in WO 2013154728 Al.

[0155] In some embodiments, used with any of the formulations described herein, an effective amount of the RSV comprises about 5.6 logio plaque forming units (PFU) per dose. In some embodiments, used with any of the formulations described herein, an effective amount of the RSV comprises about 6.2 logio plaque forming units (PFU) per dose. In certain embodiments, used with any of the formulations described herein, an effective amount of the RSV comprises about 5.6 logio PFU to 6.2 logio PFU per dose. In certain embodiments, used with any of the formulations described herein, an effective amount of the RSV comprises about 5 logio PFU to 9 logio PFU per dose. In some embodiments, used with any of the formulations described herein, an effective amount of the RSV comprises about 5 logio, 6 logio, 7 logio, 8 logio, or 9 logio PFU per dose. In some embodiments, used with any of the formulations described herein, an effective amount of the RSV comprises about 5 logio to about 8 logio PFU per dose. In some embodiments, used with any of the formulations described herein, an effective amount of the RSV comprises about 5 to about 6 logio PFU per dose or about 6.1 to about 7 logio PFU per dose or about 7.1 to about 8 logio PFU per dose or about 8.1 to about 9 logio PFU per dose.

[0156] In some embodiments, the RSV vaccine is delivered intranasally in a liquid formulation. In some embodiments, the RSV vaccine dose is delivered intranasally in a volume of about 0.2 mL. In some embodiments, the 0.2 mL dose is delivered intranasally wherein about 0.1 mL is delivered to each nostril. In some embodiments, the RSV vaccine dose is delivered intranasally using the intranasal atomization delivery device in a volume of about 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, or 1.0 mL. As described above, the dose may be divided evenly between two nostrils, divided substantially evenly between two nostrils, divided unevenly between two nostrils, or delivered all to one nostril in one or more deliveries.

[0157] In some embodiments, the virus (e.g., paramyxovirus) is a parainfluenza virus (PIV), including human (HPIV), optionally wherein the HPIV is HPIV-1, -2, -3, or -4. In someembodiments, the virus or paramyxovirus causes respiratory tract infections. In some embodiments, the PIV is a bovine PIV (BPIV). In some embodiments, the PIV is a murine PIV (MPIV). In some embodiments, the PIV and / or PIV vaccine is disclosed in US 20180312544 Al, WO 9853078 Al, WO 0104320 Al, WO 0103744 A3, WO 0142445 A3, or WO 0202605 A3, each of which is incorporated by reference in its entirety. In some vector-based formulation embodiments, the heterologous antigen is derived from PIV virus. In some vector-based formulation embodiments, the virus comprising the heterologous antigen is derived from PIV virus.

[0158] In some embodiments, the PIV is recombinant. In some embodiments, the PIV comprises one or more than one phenotype-specifying mutation(s) introduced in selected combinations into the genome or antigenome of an infectious clone to yield desired characteristics including attenuation, temperature sensitivity, cold-adaptation, small plaque size, host range restriction, etc. In some embodiments, an infectious PIV particle, e.g., a viral or subviral particle is produced from one or more isolated polynucleotide molecules encoding a PIV genome or antigenome. An infectious PIV particle may be produced by co-expressing an expression vector comprising an isolated polynucleotide molecule encoding a PIV genome or antigenome in a cell or cell-free system with an expression vector comprising one or more isolated polynucleotide molecules encoding N, P, and L proteins of a PIV. In some embodiments, the PIV or recombinant PIV is utilized as a live-attenuated vaccine (z.e., is not a vector-based vaccine).

[0159] In some embodiments, the vaccine is a PIV vector-based vaccine. In one such embodiment, the formulation comprises a recombinant human, bovine, or murine parainfluenza virus vector, optionally wherein PIV is bovine or human PIV3. In some embodiments, a PIV is used as a vector for expressing heterologous antigens (e.g., RSV, hMPV, measles virus, or mumps virus). In some embodiments, the formulation comprises a PIV vector and an RSV F antigen. In some embodiments, the formulation comprises a PIV vector and an hMPV antigen. In some embodiments, the formulation comprises a PIV vector and a human metapneumovirus (hMPV), RSV, HSV, coronavirus, Newcastle disease virus, yellow fever virus, dengue virus, or other enveloped virus antigen.

[0160] In some embodiments, the vaccine is a PIV vector-based vaccine comprising PIV3. In some embodiments, the vaccine further comprises at least one antigen from a non-PIV virus. In some embodiments, the further non-PIV antigen comprises human metapneumovirus (hMPV), RSV, HSV, coronavirus, Newcastle disease virus, yellow fever virus, dengue virus, or other enveloped viruses.

[0161] In some embodiments, the virus is a member of the Herpesviridae family. In some embodiments, the virus is a herpes simplex virus (HSV). In some embodiments, the HSV is herpes simplex virus 1 (HSV-1), Human alphaherpesvirus 1, herpes simplex virus 2 (HSV-2) or Human alphaherpesvirus 2. In some embodiments, the HSV causes blisters, small ulcers, and / or fever.

[0162] In some embodiments, the virus is a member of the Pneumoviridae family. In some embodiments, the virus is a human metapneumovirus (hMPV). In some embodiments, the hMPV causes respiratory tract infection.

[0163] In some embodiments, the virus is an influenza (flu) virus. In some embodiments, the influenza virus is influenza A, B, C, or D. In various embodiments, the influenza A virus is of the subtype H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9, or H6N1. In some embodiments, the virus is of the Yamagata lineage. In some embodiments, the virus is of the Victoria lineage. In some embodiments, an influenza antigen is incorporated in a vector-based vaccine, wherein the influenza antigen is optionally a hemagglutinin (HA) or neuraminidase (NA) protein. In some embodiments, the influenza virus causes fever, runny nose, sore throat, muscle and joint pain, headache, coughing, and / or malaise.

[0164] In some embodiments, the virus is a flavivirus. In various embodiments, the flavivirus is one of approximately 70 viruses of the Flavivirus genus of the Flaviviridae family. In some embodiments, the flavivirus is yellow fever virus, dengue virus, Japanese encephalitis virus, West Nile virus, Zika virus, and tick-borne encephalitis virus. In some embodiments, the yellow fever virus causes fever, chills, muscle pain, headache, or yellow skin. In some embodiments, the dengue virus causes fever, headache, muscle and joint pain, or rash. In some embodiments, the Japanese encephalitis virus causes headache, fever, vomiting, confusion and / or seizures. In some embodiments, the West Nile virus causes fever, headache, vomiting, neck stiffness, confusion, seizures, and / or rash. In some embodiments, the Zika virus causes fever, red eyes, joint pain, headache, and / or maculopapular rash. In some embodiments, the tick-bom encephalitis virus causes fever, malaise, headache, nausea, vomiting, and / or myalgias.

[0165] In some embodiments, the virus is a measles virus. In some embodiments, the measles virus is of the genus Morbillivirus within the family Paramyxoviridae. In some embodiments, the measles vims is measles morbillivirus (MV), rubeola vims or rubella vims. In some embodiments, the measles vims causes fever, cough, mnny nose, inflamed eyes, and / or rash.

[0166] In some embodiments, the virus is a mumps virus. In some embodiments, the mumps virus is of the genus Orthorubulavirus and the family Paramyxovirus. In some embodiments, the mumps virus is Mumps orthorubulavirus or Mumps rubulavirus. In some embodiments, the mumps virus causes fever, muscle pain, headache, and / or painful swelling of the parotid gland.

[0167] In some embodiments, the virus is a rubella virus. In some embodiments, the rubella virus is a togavirus. In some embodiments, the rubella virus causes fever, swollen lymph nodes, malaise, and / or rash.

[0168] In some embodiments, the virus is a chickenpox virus. In some embodiments, the chicken pox virus is the varicella zoster virus. In some embodiments, the chickenpox virus causes small, itchy blisters, fever, tiredness and / or headaches.

[0169] In some embodiments, the virus is a smallpox virus. In some embodiments, the smallpox virus is Variola major or Variola minor. In some embodiments, the smallpox virus causes fever, vomiting, sores in the mouth, and / or a skin rash that turns into characteristic fluid- filled bumps with a dent in the center, wherein the bumps scab over and fall off, leaving scars.

[0170] In some embodiments, the virus is a poliovirus. In some embodiments, the poliovirus is of the species Enterovirus C in the family of Picornaviridae. In some embodiments, the poliovirus causes abortive poliomyelitis. In various embodiments, the poliovirus causes paralytic or nonparalytic polio.

[0171] In some embodiments, the virus is a rotavirus. In some embodiments, the rotavirus is of the family Reoviridae. In some embodiments, the rotavirus is Rotavirus A, B, C, D, E, F, G, H, I, or J. In some embodiments, the rotavirus causes nausea, vomiting, watery diarrhea, and / or low-grade fever.

[0172] C. Adjuvants

[0173] In some embodiments, the vaccine comprises an adjuvant. Any adjuvant known to those of skill in the art is encompassed. Non-limiting exemplary adjuvants include, incomplete Freund’s adjuvant, aluminum phosphate, aluminum hydroxide, alum, Stimulon™ QS-21 (Aquila Biopharmaceuticals, Inc., Worchester, Mass.), MPL™ (3-0-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamilton, Mont.), and interleukin- 12 (Genetics Institute, Cambridge, Mass.).

[0174] In some embodiments, the formulation is liquid. In some embodiments, the formulation is dried, e.g., lyophilized (lyo).III. Articles of Manufacture

[0175] In some embodiments, the formulation is provided in a container, device, or kit. In one embodiment, the device is used for parenteral, e.g., intranasal, intramuscular, subcutaneous delivery, or by inhalation. In some embodiments, the formulation is provided in a syringe (such as pre-filled syringe), automatic injector, intranasal device, such as a syringe, atomization delivery device (e.g., the formulation is atomized), dropper, or injection device. Such pre-filled syringe or autoinjector device comprises a formulation that can maintain a titer loss under 1 logio PFU / ml at room temperature for at least 3-6 months, and at 2-8 degrees Celsius for at least 12-24 months.

[0176] In some embodiments, the formulation is provided in a container, which includes, but is not limited to, a syringe (including pre-filled syringe), automatic injector, intranasal atomization delivery device, or bottle. A pre-filled device for intranasal administration (e.g., spray or aerosolized container) comprising the formulation is encompassed. In some embodiments, the pre-filled intranasal device is formulated for direct administration to a subject, optionally a pediatric subject. Such pre-filled device for intranasal administration comprises a formulation that can maintain a titer loss under 1 logio PFU / ml at room temperature for at least 3, 4, 5, or 6 months, and at 2-8 degrees Celsius for at least 12-24 months.IV. Methods of Using and Uses

[0177] In some embodiments, a method of preventing or reducing the likelihood of infection by an enveloped RNA or DNA virus in a subject is provided comprising administering a formulation as disclosed herein. In some embodiments, a method of vaccinating a subject against infection by an enveloped RNA or DNA virus is provided comprising administering a formulation as disclosed herein. In some embodiments, methods of triggering or generating an immune response in a subject are provided comprising administering the formulation of the disclosure.

[0178] In some embodiments, the formulation is for use in preventing or reducing the likelihood of infection by an enveloped RNA or DNA virus in a subj ect. In some embodiments, the formulation is for use in a method of vaccinating a subj ect against infection by an enveloped RNA or DNA virus. In some embodiments, the formulation is for use in triggering or generating an immune response in a subject.

[0179] In some embodiments, use of the formulation for the manufacture of a medicament for preventing or reducing the likelihood of infection by an enveloped RNA or DNA virus in a subject is provided. In some embodiments, use of the formulation for the manufacture of amedicament for vaccinating a subject against infection by an enveloped RNA or DNA virus is provided. In some embodiments, use of the formulation for the manufacture of a medicament for triggering or generating an immune response in a subject is provided.

[0180] In each of the method and use embodiments provided herein, infection may be caused by any one of the viruses described above.

[0181] In some embodiments, the method and use is for treating an infection caused by a virus of the Paramyxoviridae family. In some embodiments, the paramyxovirus is a respiratory syncytial virus (RSV). In some embodiments, the RSV is human RSV (also known as Human orthopneumovirus). In some embodiments, the virus is RSV and comprises a formulation for administration to pediatric subjects (RSV ANS2 / A1313 / 11314L) as disclosed, for example, in WO 2017100756 Al, which is incorporated by reference in its entirety. In some embodiments, the virus is RSV and comprises an attenuated RSV with point mutations, as described, for example, in WO 2013154728 Al, which is incorporated by reference in its entirety. In some embodiments, the virus is RSV and comprises mutations in the RSV open reading frames that interfere with RSV protein expression, as described, for example, in WO 2017100759 Al, which is incorporated by reference in its entirety. In some vector-based formulation embodiments, the heterologous antigen is derived from an RSV virus, optionally wherein the antigen is RSV F and / or RSV G. In some embodiments, the virus is RSV and comprises a formulation for administration to pediatric subjects (RSV ANS2 / A1313 / 11314L) that is a recombinant infectious respiratory syncytial virus including a large polymerase protein (L), phosphoprotein (P), nucleocapsid protein (N), an M2-1 protein nonstructural protein 1 (NS1), a glycoprotein (G), a fusion protein (F), a matrix protein (M), an M2-2 protein, and a small hydrophobic protein (SH), and a genome or antigenome having: a deletion of the codon that encodes the serine at position 1313, or a corresponding position, of the L protein, a mutation of amino acid sequence residue 1314, or a corresponding position, of the L protein, wherein the mutation of L protein amino acid sequence residue 1314 is an amino acid substitution of leucine for isoleucine, wherein the leucine is encoded by a codon set forth as CTG and a deletion of the NS2 gene, as described, for example, in WO 2013 / 154728 Al.

[0182] In some embodiments, the method and use are for treating an infection caused by a parainfluenza virus (PIV), including human PIV (HPIV), optionally wherein the HPIV is HPIV-1, -2, -3, or -4. In some embodiments, the PIV causes respiratory tract infections. In some embodiments, the PIV is a bovine PIV (BPIV). In some embodiments, the PIV is a murine PIV (MPIV). In some embodiments, the PIV and / or PIV vaccine is disclosed in US20180312544 Al, WO 9853078 Al, WO 0104320 Al, WO 0103744 A3, WO 0142445 A3, or WO 0202605 A3, each of which is incorporated by reference in its entirety.

[0183] In some embodiments, the method and use is for treating an infection caused by a virus of the Herpesviridae family. In some embodiments, the virus is a herpes simplex virus (HSV). In some embodiments, the HSV is herpes simplex virus 1 (HSV-1), Human alphaherpesvirus 1, herpes simplex virus 2 (HSV-2), or Human alphaherpesvirus 2. In some embodiments, the HSV causes blisters, small ulcers, and / or fever.

[0184] In some embodiments, the method and use is for treating an infection caused by a virus of the Pneumoviridae family. In some embodiments, the virus is a human metapneumovirus (hMPV). In some embodiments, the hMPV causes respiratory tract infection.

[0185] In some embodiments, the method and use is for treating an infection caused by an influenza (flu) virus. In some embodiments, the influenza virus is influenza A, B, C, or D. In various embodiments, the influenza A virus is of the subtype H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9, or H6N1. In some embodiments, the virus is of the Yamagata lineage. In some embodiments, the virus is of the Victoria lineage. In some embodiments, the influenza virus causes fever, runny nose, sore throat, muscle and joint pain, headache, coughing, and / or malaise.

[0186] In some embodiments, the method and use is for treating an infection caused by a flavivirus. In various embodiments, the flavivirus is one of approximately 70 viruses of the Flavivirus genus of the Flaviviridae family. In some embodiments, the flavivirus is yellow fever virus, dengue virus, Japanese encephalitis virus, West Nile virus, Zika virus, and tick- borne encephalitis virus. In some embodiments, the yellow fever virus causes fever, chills, muscle pain, headache, or yellow skin. In some embodiments, the dengue virus causes fever, headache, muscle and joint pain, or rash. In some embodiments, the Japanese encephalitis virus causes headache, fever, vomiting, confusion, and / or seizures. In some embodiments, the West Nile virus causes fever, headache, vomiting, neck stiffness, confusion, seizures, and / or rash. In some embodiments, the Zika virus causes fever, red eyes, joint pain, headache, and / or maculopapular rash. In some embodiments, the tick-bom encephalitis virus causes fever, malaise, headache, nausea, vomiting, and / or myalgias.

[0187] In some embodiments, the method and use is for treating an infection caused by a measles virus. In some embodiments, the measles vims is of the Qn\isMorbillivirus within the family Paramyxoviridae. In some embodiments, the measles vims is measles morbillivirus(MV), rubeola virus, or rubella virus. In some embodiments, the measles virus causes fever, cough, runny nose, inflamed eyes, and / or rash.

[0188] In some embodiments, the method and use is for treating an infection caused by a mumps virus. In some embodiments, the mumps virus is of the genus Orthorubulavirus and the family Paramyxovirus. In some embodiments, the mumps virus is Mumps orthorubulavirus or Mumps rubulavirus. In some embodiments, the mumps virus causes fever, muscle pain, headache, and / or painful swelling of the parotid gland.

[0189] In some embodiments, the method and use is for treating an infection caused by a rubella virus. In some embodiments, the rubella virus is a togavirus. In some embodiments, the rubella virus causes fever, swollen lymph nodes, malaise, and / or rash.

[0190] In some embodiments, the method and use is for treating an infection caused by a chickenpox virus. In some embodiments, the chicken pox virus is the varicella zoster virus. In some embodiments, the chickenpox virus causes small, itchy blisters, fever, tiredness, and / or headaches.

[0191] In some embodiments, the method and use is for treating an infection caused a smallpox virus. In some embodiments, the smallpox virus is Variola major or Variola minor. In some embodiments, the smallpox virus causes fever, vomiting, sores in the mouth, and / or a skin rash that turns into characteristic fluid-filled bumps with a dent in the center, wherein the bumps scab over and fall off, leaving scars.

[0192] In some embodiments, the method and use is for treating an infection caused by a poliovirus. In some embodiments, the poliovirus is of the species Enterovirus C in the family of Picornaviridae. In some embodiments, the poliovirus causes abortive poliomyelitis. In various embodiments, the poliovirus causes paralytic or nonparalytic polio.

[0193] In some embodiments, the method and use is for treating an infection caused by a rotavirus. In some embodiments, the rotavirus is of the family Reoviridae. In some embodiments, the rotavirus is Rotavirus A, B, C, D, E, F, G, H, I, or J. In some embodiments, the rotavirus causes nausea, vomiting, watery diarrhea, and / or low-grade fever.

[0194] Formulations of the present disclosure may be used alone at appropriate dosages which allow for optimal inhibition of viral infection with minimal potential toxicity. In addition, co-administration or sequential administration of other agents may be desirable.

[0195] The formulations and compositions of the present invention may be administered to a patient intranasally, by intramuscular injection, by subcutaneous injection, by intradermal introduction, or impression though the skin. Other modes of administration such as intraperitoneal, intravenous, or inhalation delivery are also contemplated. In someembodiments, formulations and compositions of the present disclosure may be delivered by intranasal atomization. In some embodiments, the formulation is administered to a subject directly, e.g., without reconstitution or dilution.

[0196] In some embodiments, the pharmaceutical compositions and formulations disclosed herein are administered to a patient in various prime / boost combinations in order to induce an enhanced, durable immune response. In this case, two pharmaceutical compositions are administered in a “prime and boost” regimen. For example, the first composition is administered one or more times, then after a predetermined amount of time (for example, 2 weeks, 1 month, 2 months, six months, or other appropriate interval) a second composition is administered one or more times.

[0197] In some embodiments, the live-attenuated viral vaccine is administered by injection. In some embodiments, the live-attenuated viral vaccine is in the form of a nasal spray. In some embodiments, the live-attenuated viral vaccine is administered in the form of an atomized intranasal spray. In some embodiments, the live-attenuated viral vaccine is an oral vaccine. In various embodiments, the vaccine is administered in one or more doses.

[0198] In any of the foregoing embodiments involving a subject, the subject can be mammalian. In any of the foregoing embodiments involving a subject, the subject can be human. In any of the foregoing embodiments involving a subject, the subject can be a cow, pig, monkey, sheep, dog, cat, fish, or poultry.EXAMPLES

[0199] The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way.

[0200] Example 1. Materials and Methods

[0201] 1.1. Materials

[0202] The RSV vaccine strain, RSV ANS2 / A1313 / 11314L, is a live-attenuated virus vaccine genetically designed to contain a whole gene deletion (ANS2) and point mutations (A1313 and 11314L) to achieve a suitable level of attenuation while maintaining an effective level of immunogenicity via intranasal administration to induce innate, humoral, and cell- mediated responses at mucosal surfaces.Table 1: Active Material ListStudy # DS lot # and starting Example Table Figure compositions in _ purification buffer* _ i*Drug product formulations tested in examples below are made from active material listed in Table 1.Table 2: Chemical Material List

[0203] Example 1.2. Methods

[0204] 1.2.1. Stability Assessment

[0205] An accelerated temperature stability study was designed to assess formulations of samples that were stored at 37 °C ±2 °C for up to 2 or 3 weeks. In addition, data was collected for samples stored at 25 °C ±2 °C up to 8 or 12 weeks and 45 °C ±2 °C up to 5 or 8 days. “Accelerated” in this context means that it was run above storage temperature of about 2-8 °C such that the reaction or degradation would likely be accelerated compared to the storage temperature.

[0206] Real-time stability studies used storage conditions of 2 °C to 8 °C for up to 12 months, 24 months, 36 months, or longer.

[0207] Freeze-Thaw (F / T) stress: A freeze-thawing stress study was designed and incorporated 5 freeze-thaw cycles between below -60 °C and room temperature. All 5 samples were assessed together with a frozen control at time zero (TO).

[0208] The bioactivity of the samples in each of the stability assessment studies was assessed by plaque assay as described below (a stability indicating assay that can directly measure virus infectious titer of each stability time point).

[0209] 1.2.2. Freeze-Drying (FD)

[0210] Freeze drying (FD) (also sometimes referred to herein as “lyophilizing”, “lyo”, or the like) is a process used to remove water from a formulation at low temperatures and prevent thermal degradation through a process of sublimation. There are three phases involved in the present FD process: freezing, primary drying, and secondary drying. Samples are all subject to a pre-cooling step at 4 °C so that the samples are at a uniform starting temperature prior to freeze drying (lyophilization). In this study, formulations were lyophilized (lyo) in the FTS LyoStar II freeze dryer. The drying parameters are provided in Table 3.Table 3 : Example of FD Cycle Parameters

[0211] 1.2.3 Spray Drying

[0212] Spray Drying (SD) is a process of converting a liquid formulation into a final product of dried powder by removing the moisture content. In SD, a liquid product is sprayed through a nozzle into a chamber containing hot air, which evaporates the solvent of the liquid formulation into a powdered form as the droplets of the liquid formulation come into contact with the hot air. The liquid formulations herein were processed with a BUCEH Mini Spray Dryer B-290 following the operating procedure. All SD components were autoclaved prior to use to allow for aseptic operation. Testing was done using the drying parameters in Table 4.Table 4: Example of SD Process Parameters

[0213] 1.2.4 Foam Drying

[0214] Foam drying is an alternative to freeze drying. Foam drying is conducted in an FTS LyoStar II Freeze dryer without a freezing phase. Dried product produced by foam drying does not form a powder cake, rather a bubbled, glassy foam is produced. Exemplary foam-drying cycle parameters are included in Table 5.Table 5: Example of Foam-Drying Cycle Parameters

[0215] 1.2.5. Infectious Titer Measurement (Plaque Assay)

[0216] The virus infectious titer was determined by plaque assay, which is an indicating assay for monitoring viral infectivity of a sample’s biological stability. A higher titer indicates that a sample has better stability or a lower titer loss of the sample indicates that a sample has better stability. There is no other stability indicating assay that has been identified for use in measuring respiratory syncytial virus (RSV) biological stability. Plaque assay is used for formulation and stability evaluation as indicated herein.

[0217] Infectious titers of RSV samples were assessed by titration on the complementing Vero cell line. 48-well plates were seeded one day prior to infection. Samples were serially diluted, plated, and incubated at 34 °C and 5% CO2 for 4 days. RSV plaques were visualized by immunostaining. Immunostaining was performed by adding conjugate primary anti-RSV antibody (5353C75.1) (1 : 1000 dilution) to each well and incubating the 48-well plates at 36 °C and 5% CO2 for 20 minutes and with 1-Step TMB-blotting solution (Thermo Fisher, USA). After washing, the plaques were counted automatically by a Viruscope (MicroVision Instruments, EVRY Cedex France). Infectious titer was then calculated by averaging oftriplicates wells and expressed as plaque forming units per milliliter (PFU / mL) or logio PFU / mL. Variation of the assay was within ±0.2 logio PFU / mL.

[0218] 1.2.6. Data Analysis

[0219] 1) Lyo Loss - Lyo loss value was calculated as the difference between pre-lyo average log titer and post-lyo average log titer.

[0220] 2) Storage Loss - Storage loss value was calculated as the difference between the average log titer value at time zero and the average log titer value after different time points and storage conditions.

[0221] 3) Data Analysis of Virus Degradation Rate - A commercial graphing software was used to trend the force degradation profile and for regression analysis. The slope, or constant of the regression equation, is considered the degradation rate. Degradation rate (i.e., the slope obtained from linear regression analysis) was used to compare formulation stability and to extrapolate the predicted degradation time at 37 °C at 14 days. The rate and time of the degradation were used for guiding selection of a stable formulation.

[0222] 4) Analysis of Covariance (ANOVA) - Drug product (DP) formulation degradation slope ANOVA analysis was done for multiple lots in the development studies for both high dose and low dose formulations. Representative drug substance (DS) lots formulated at high dose and low dose in the confirmation studies were measured to investigate the significance of differences between lots and formulations for assisting in decision making. / ?-value < 0.05 was considered as statistically significant.

[0223] 5) Advanced Kinetics and Technology Solutions (AKTS) Analysis - AKTS is software that is able to analyze kinetic models independent of reaction complexity from a one- step reaction to multi-step reactions. AKTS uses a combination of two Sestak-Berggren (SB) models with the inclusion of Arrhenius equation. The Arrhenius contribution to the model allows analysis of degradation data obtained at various storage temperatures (e.g., 5 °C, 25 °C, 37 °C, 45 °C, etc.) as opposed to traditional linear ICH modelling methods that are based only on analysis of degradation data at the normal storage condition of the product at 5 °C (ICH Guideline Q1E). AKTS simultaneous combination of the SB equation enables consideration for all kinetic models used in the literature and ranks models applied to autocatalytic type reactions. AKTS provides Akaike’s and Bayesian Information Criterion, AIC and BIC, to automatically help prevent over-fitting and discriminate the best kinetic reaction model among all mathematically fitted models. AKTS utilizes the bootstrap method to calculate confidence intervals (CI) for the predictions (e.g., 95% CI), allowing for reliable estimation of long-term stability or shelf-life prediction (see, e.g., D. Clenet et al., Advanced Kinetic Analysis as a Toolfor Formulation Development and Prediction of Vaccine Stability, J. PHARM. SCI. 103:3055- 3064, 2014). Here, 3-month data analysis was used for formulation selection purposes.

[0224] 1.2.7. Study Design

[0225] Stabilizing a live-attenuated enveloped virus can be challenging. Accordingly, many live-attenuated viral vaccine candidates (e.g., herpes simplex virus-2 (HSV-2), ALVAC, etc.) are developed as frozen liquid, and currently there is rarely success in developing a thermostable liquid live enveloped viral vaccine. The gold standard for industry is generally to freeze dry liable viruses, such as RSV, measles, yellow fever, and dengue fever. It could be advantageous (e.g., commercially advantageous) to formulate enveloped viruses to withstand long-term storage in liquid. It could also be advantageous to lengthen the time that enveloped viruses can be stored as lyophilized formulations.

[0226] One reason for developing more stable liquid and lyo formulations is due to the complexity and cost of cold chain logistics and the challenge of industrialization of frozen liquid formulations. “RSV ANS2 / A1313 / 11314L,” sometimes referred to as “RSV delta NS2” (as described in WO 2013154728), was assessed for improved storage as liquid and lyophilized forms. RSV ANS2 / A1313 / 11314L with different stabilizers in liquid and lyo formulations were first assessed at accelerated temperature, 37 °C ±2 °C. The potential stable formulations were then further studied under real time storage conditions at 5 °C ±3 °C for up to 3 months, 6 months, 12 months, 24 months, or 36 months.

[0227] 1.2.8. Criteria for Formulation Selection

[0228] The selection criteria for a formulation was infectious titer loss. In general, the smaller the titer loss during formulation processing and storage, the better the stability of the formulation.

[0229] World Health Organization (WHO) has set guidelines on the evaluation of vaccine stability and recommends conducting basic and accelerated stability studies (Guidelines on the Stability Evaluation of Vaccines, Geneva: WorldHealth Organization,' 2006. 28 p. Report No.: WHO / BS / 06.2049 Final) (WHO Technical Report Series No. 962: Guidelines on the Stability Evaluation of Vaccines, Geneva: World Health Organization,' 2011. 28. Report No.: 57). However, different criteria have been used in the literature and different titer losses for different live-attenuated viral formulations have been reported. For example, less than 1 logio PFU / mL loss up to 6 weeks at 25 °C was used for an influenza vaccine (White JA et al., Vaccine, 2016;34(32):3676-83), which provided storage stability for up to one year at 2 °C to 8 °C. In another example, less than 1 logio PFU / mL for 8 hours at 40 °C was used for measles formulations (Schlehuber et al., Vaccine, 2011;29(31): 5031-9). In general, a common criteriondescribed in the literature is a titer loss of less than 1 logio PFU / mL for 1 week at 37 °C (Ohtake5 et al., Vaccine; 2010;28(5): 1275-84; Wiggan et al., Vaccine, 2011;29(43):7456-62; and Bhambhabi A et al., US Patent Application Publication No. 2016 / 0250319).

[0230] For these studies, we aimed to find a formulation that would allow a titer loss of less than 1 logio PFU / mL for 1-2 weeks, e.g., less than 1 logio PFU / mL at 10 days or 2 weeks at 37 °C (Ohtake S et al., Vaccine; 2010;28(5): 1275-84).

[0231] Accelerated stability data at 37 °C was used for formulation selection with a few real-time stability time points because the accelerated stability test is convenient and time saving. In confirmation studies, virus stability was also monitored at different accelerated storage temperatures at 25 °C and 45 °C. The test samples had significant and readily detectable degradations in a relatively short period of time. The degradation rates of each formulation at 37 °C were compared for formulation selection. The freeze-thawing stress study was designed and incorporated 5 freeze-thaw cycles between < -60 °C and room temperature.

[0232] Example 2. Excipient Screening Studies for Liquid and Lyo Formulations

[0233] A panel of stabilizers and buffers at different pH values was scanned to understand the function and effects of the stabilizers and buffers during formulation. Both liquid and lyo formulations were examined after the screening of excipients.

[0234] 2.1. Effect of Trehalose and Salt Concentrations

[0235] Formulation studies in Study 1 were conducted at different trehalose and salt concentrations starting with drug substance (DS) LI, and adjusting as needed for the study by dilution or addition of components. Four excipients and their combinations were studied for both liquid and lyo formulations. The liquid and lyo formulations were assessed according to the experimental design depicted in FIG. 1A (key: trehalose (10% = -1, 20% = 0, and 30% = 1), NaCl (0 mM = -1, 80 mM = 0, and 160 mM = 1), monosodium glutamate (MSG) (0 mM = -1, 50 mM = 0, and 100 mM = 1), histidine (His) (3.33 mM = -1, 10 mM = 0, and 16.67 mM = 1). Results of the plaque assay for all formulation compositions in FIG. 1A are shown in Table6 and FIGs. IB and 1C. Table 6 includes the comparison based on the accelerated thermal stability performance of the different formulations.Table 6: Summary of Excipient Concentration Impact on StabilityNote: T = trehalose; N=NaCl; M=MSG; H=histidine

[0236] FIG. IB depicts the dose effects observed and shows that trehalose is a key stabilizer for RSV ANS2 / A1313 / 11314L in liquid formulations (see black bars). Significantly, titer loss observed in 10% trehalose formulations (see horizontal arrows labeled 10%, 20%, and 30%), except F5 (30% trehalose without MSG and NaCl) and F10 (30% trehalose without NaCl) in liquid, have greater titer loss compared to titer loss observed in 20% and 30% trehaloseformulations. Storage loss is about or <1 logio PFU / mL at 37 °C for 1 week in most 30% trehalose formulations that contain salts. As depicted in FIG. IB and Table 6, it is also suggested that 10% trehalose formulations have a reduced protective effect in the liquid formulations compared to 20% and 30% trehalose formulations.

[0237] Additionally, FIG. IB shows trehalose concentration is also a key stabilizer for lyo formulations (see white bars). 30% trehalose formulations had the least titer loss in both the lyophilization process (e.g., manufacturing) and storage (e.g., shelf life) compared to 20% trehalose and 10% trehalose formulations (see arrows labeled 10%, 20%, and 30%). 20% trehalose lyo formulations also showed decreased titer loss compared to 10% trehalose lyo formulations.

[0238] As indicated in FIG. 1C, NaCl can be a stabilizer for 30% trehalose liquid formulations. For example, formulations Fl and F8, which contain NaCl, had better stability than formulations F 10 and F5, which lack NaCl. In addition, formulation F5, which lacks both monovalent salts NaCl and MSG, has an infectious titer loss of 3 loglO PFU / mL after 1 week at 37 °C even with 30% trehalose and histidine.

[0239] It was determined that both liquid and lyo 30% trehalose formulations were able to retain biological activity after 2 weeks of incubation at 37 °C. Furthermore, in addition to 30% trehalose, samples containing NaCl have a better liquid stability profile indicating that NaCl improves liquid stability. Samples including 30% trehalose and MSG with or without NaCl also had improved liquid stability. Therefore, 30% trehalose NaCl and / or MSG effectively preserved viruses and improved thermal stability in both liquid and lyo formulations. Among the excipients tested, trehalose was the only excipient assessed that significantly impacted stability profile. In addition, monovalent salt had a synergetic effect with trehalose.

[0240] Formulations with low (10%) trehalose content formulations were not stable in both liquid and lyo.

[0241] 2.2. 40% Trehalose Liquid Formulation Concentration Study

[0242] To determine if a higher percentage of trehalose can further improve liquid formulation stability, a study (Study 2) was performed to compare a 30% trehalose control (Fl Control) with 40% trehalose (F2). A development lot L2 of RSV ANS2 / A1313 / 11314L (in 30% or 40% trehalose, 10 mM histidine, 160 mM NaCl, and 100 mM MSG at pH 7) was used in this study. Table 7 summarizes stability data at different storage temperatures. The results demonstrated that 30% trehalose formulations were stable up to 12 months in a 5 °C storage conditions with about 0.6 logio PFU / mL loss. 40% trehalose formulations (F2) provided even better stability with less titer loss (only about 0.5 logio PFU / mL) at 12 months in a 5 °C storagecondition. Hence, 40% trehalose formulation has better stability performance compared to 30% trehalose formulation under these conditions.Table 7: Stability Data Summary of Study 2

[0243] 2.3. 15% Trehalose Liquid Formulation Concentration Study

[0244] A study (Study 3) was designed to assess the short-term stability performance of a 15% trehalose formulation compared to a 30% trehalose formulation, both 15% and 30% formulations included 160 mM NaCl, 100 mM MSG, and 10 mM histidine. This study was used to determine drug product (DP) formulation (L3 and L4) and the mixing or dilution strategy for an adjuvant or co-administration with other antigens if required. After a period of14 days, the titer loss exceeded the 1.0 logio PFU / mL limit discussed above and the 15% trehalose concentration was not used for further formulations.

[0245] FIG. 2A summarizes thermal stability profiles of 15%, 20%, 30%, and 40% trehalose formulations having the same salt / buffer composition in each formulation, depicting a comparison of decreasing infectious titer trends over a period up to 21 days at 37 °C.

[0246] FIG. 2B summarizes the stability profile of 15%, 30%, and 40% trehalose formulations, depicting a comparison of decreasing infectious titer trends over a period up to 12 months at 5 °C.

[0247] For these comparisons, it was concluded that formulations with 30% and 40% trehalose had superior stability performance to formulations with 15% and 20% trehalose.

[0248] Example 3. Stable 30% Trehalose Liquid Formulation of RSV ANS2 / A1313 / I1314L

[0249] Based on the data described above, a formulation including 30% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0 was selected for testing. Stability studies were conducted for the RSV ANS2 / A1313 / 11314L. 3 mL clear glass vials with rubber stoppers and aluminum caps were filled with 0.6 mL of the vaccine product. The studies described below demonstrated long-term, real-time stability of RSV ANS2 / A1313 / 11314L in this liquid formulation at 5 °C.

[0250] 3.1 Real Time Stability Study of Two Preparations of RSVANS2 / A1313 / I1314L

[0251] For RSV ANS2 / A1313 / 11314L (L5) (in 30% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0) the stability results obtained from a development stability study (Study 4) confirmed a shelf-life period of 36 months at < -60 °C from the date of vial filling. In addition, FIG. 3 A shows that there was only 0.79 logio PFU / mL titer loss after storage for 24 months at 5 °C in liquid formulation.

[0252] For a clinical DP RSV ANS2 / A1313 / 11314L (L6) in 30% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0 the stability results obtained for a development stability study (Study 5) confirmed a shelf-life period of 24 months at < -60 °C from the date of vial filling. In addition, FIG. 3B shows that there was only 0.87 logio PFU / mL titer loss after storage for 24 months at 5 °C in liquid formulation.

[0253] These studies provided evidence that the 30% and 40% trehalose formulations with combinations of salts and histidine at pH 7.0, as described herein, are stable liquid viral formulations.

[0254] 3.2. Stability Study of an RSV ANS2 / A1313 / I1314L (DS L5)

[0255] Purified drug substance (DS) of RSV ANS2 / A1313 / 11314L (L5) was stabilized in the same formulation composition as provided above of 30% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0. The stability data of this DS L5, which was obtained from 120 L DS fermentation and purification processed lot, was subjected to a 5 cycle freezethaw (see FIG. 4A). The results of a study 6 using RSV ANS2 / A1313 / 11314L (L5) thawed from -60 °C and stored at 5 °C for up to 3 months is shown in FIG. 4B. The results of study 6 using RSV ANS2 / A1313 / 11314L (L5) thawed from -60 °C and stored at 25 °C for up to 5 weeks is shown in FIG. 4C. These results suggest RSV ANS2 / A1313 / 11314L in 30% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0 is a stable formulation and suitable for industrial manufacturing implementation.

[0256] Example 4. Stable Liquid Formulation in 40% Trehalose

[0257] Example 4, as further described below, demonstrates that a formulation composition with a higher concentration of trehalose, specifically, 40% trehalose, 100 mM MSG, 160 mM NaCl, and 10 mM histidine at pH 7.0, can further minimize the titer loss of the liquid DP formulation during short-term and long-term storage. This supports use of the formulation, for example, in commercial use as a stable liquid product stored at 5 °C in a classic glass vial or pre-filled syringe.

[0258] 4.1 Stability Study of 40% Trehalose Liquid Formulation of RSVANS2 / A1313 / I1314L (Study 7)

[0259] This study confirmed 40% trehalose liquid formulation stability at high dose (HD, 6.7 logio PFU / mL) and low dose (LD, 5.7 logio PFU / mL) of RSV ANS2 / A1313 / 11314L at various incubation temperatures. Two DS lots L5 and L7 were used in this study. Each RSV ANS2 / A1313 / 11314L DS lot had the same composition of 40 % trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0.

[0260] RSV ANS2 / A1313 / 11314L in liquid formulations of 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0 (DP formulations) were monitored at 4 different temperatures including real time storage conditions at 5 ±3 °C for up to 24 months. The incubation temperatures for accelerated stability testing were 25 ±2 °C, 37 ±2 °C, and 45 ±2 °C. Infectious titer results for 40% trehalose formulations at real time storage conditions at 5 ±3 ° are shown in FIGs. 5A (high dose, e.g., about 6 logio PFU / dose) and 5B (low dose, e.g., about 5 logio PFU / dose). Stability data is shown in Tables 8-11.Table 8: Liquid Formulation 40% Trehalose (High Dose of RSV ANS2 / A1313 / I1314L) Development Lots Accelerated Stability at 37 °CTable 9: Liquid Formulation 40% Trehalose (Low Dose (LD) of RSV ANS2 / A1313 / I1314L) Development Lots Accelerated Stability at 37 °CTable 10: Liquid Formulation 40% Trehalose (High Dose (HD) of RSV ANS2 / A1313 / I1314L) Development Lots Real Time Stability at 2 °C to 8 °CTable 11: Liquid Formulation 40% Trehalose (Low Dose (LD) of RSV ANS2 / A1313 / I1314L) Development Lots Real Time Stability at 2 °C to 8 °C

[0261] The above results indicated that 40% trehalose is a stable liquid formulation that reaches no more than 1 logio PFU / mL titer loss after storage for 36 months at 5 °C in a longterm stability study. In addition, no more than 1 logio PFU / mL titer loss after storage for 7 days at 37 °C was observed.

[0262] 4.2. Stability Study of DS Lot 8 (Study 8)

[0263] In light of the stability results above, purified DS RSV ANS2 / A1313 / 11314L L8 was stabilized in a formulation composition including 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0 for further industrial scale-up and in an attempt to achieve a more stable liquid product. A DS lot 8 of RSV ANS2 / A1313 / 11314L was freeze-thawed five times and stability data was measured as depicted in FIG. 6A. The results of study 8 using RSV ANS2 / A1313 / 11314L (L8) thawed from -60 °C and stored at 5 °C up to 3 months is shown in FIG. 6B, up to 24 months is shown in Fig. 6C where the titer loss is 1.31 log, and stored at 25 °C for up to 5 weeks is shown in FIG. 6D. Results suggested that 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0 is a stable formulation suitable for commercial manufacturing implementation.

[0264] 4.5. Stability Study of a DP Lot and AKTS Prediction (Study 9)

[0265] A development study was performed to document stability performance of a 2 L scale-up DP batch of RSV ANS2 / A1313 / 11314L (L9) in 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0 formulation, and to predict the DP’s shelf-life byAKTS at a 3-month timepoint of liquid product stored at 5 ±3 °C and accelerated conditions (25 ±2 °C, 37 ±2 °C, and 45 ±2 °C). The 2L DP batch was made from a 200 L processed DS Lot 9. 3 mL clear glass vials with rubber stoppers and aluminum caps were filled with 0.6 mL samples of the DP batch. The plaque assay described above was used for measuring sample infectious titer at all stability time points.

[0266] The development stability study data and product shelf-life prediction are summarized in FIGs. 7A-F. FIG. 7A shows the stability profile at a storage condition of 2-8 °C for up to 24 weeks; FIG. 7B shows the stability profile at a storage condition of 2-8 °C for up to 24 months ; FIG. 7C shows the stability profile at an accelerated storage condition of 25 °C for up to 9 weeks; and FIG. 7D shows the stability profile at forced degradation conditions of 37 °C for up to 21 days. FIG. 7E shows the stability profile at forced degradation conditions of 45 °C for up to 5 days. FIG. 7F shows the formulated bulk product (FBP) stability profile at 5 °C for up to 3 months, which is stable without titer loss. More details of the FBP stability study are described in the section 4.6. The 3-month stability data was analyzed by the AKTS program to predict 24-month shelf life (SL) at 5 °C (FIG. 7G). The shelf-life prediction by AKTS is summarized in Table 12. FIG. 7B shows the actual titer loss at 24 months at 5 °C is 0.785 log.

[0267] 4.6. Holding Time Study of Formulated Bulk Product (FBP)

[0268] FBP formulation is one phase of the vaccine manufacturing process that utilizes calculations to generate ingredient volume requirements based on known values, variables, and formulas of formulation parameters (i.e., no serial dilution required). Ingredient quantities are calculated based on desired batch size of the FBP. The amount of each ingredient (or component) to be dispensed is based on weight. Specific gravity (SG) value for DS is required at the time of preparation of the formulation to convert the active ingredient addition volume to a weight that can be measured on a scale. If required, samples of 40% trehalose, 160 mM NaCl, 100 mm MSG, and 10 mM histidine at pH 7.0 formulation buffer and DS can be taken. Here, FBP was transferred into a 250 mL Nalgene bottle. The 200 mL FBP was held at 2-8 °C and samples were taken at 1-day, 2-day, 3-day, 1-month, 2-month, and 3-month timepoints. As shown in FIG. 7E, the results suggested no significant titer loss over a 3-month period.

[0269] 4.7. Comparability between DP Lots from Different DS Lots

[0270] The stability performance of a formulation including 40% trehalose, 160 mMNaCl, 100 mM MSG, and 10 mM histidine at pH 7.0 was further assessed. The stability performance comparison of two DP lots was performed to assess the stability profile at 37 °C and the stability profile at 5 °C. One DP lot was made from a 120L process RSV ANS2 / A1313 / 11314L(L7) and one DP lot was made from a 200-L process DS lot RSV ANS2 / A1313 / 11314L (L9). FIG. 8A depicts a thermal stability comparison at 37 °C between DS L7 and DS L9 ( / ?-value is 0.2682 at 21 days). FIG. 8B depicts a thermal stability comparison at 5 °C between DS L7 and DS L9 ( / ?-value is 0.8850 at 6 month). FIG. 8C depicts a thermal stability comparison at 5 °C between DS L7 and DS L9 (at 24 months). FIG. 8D is an AKTS prediction comparison (see also Table 12).Table 12: AKTS SL-Prediction Based on 3-Month Stability Data* Estimated values based on AKTS model output for information only due to insufficient data

[0271] The above data and comparison indicate that the 40% trehalose, 160 mM NaCl, 100 mm MSG, and 10 mM histidine at pH 7.0 formulation is a stable liquid formulation where the stability data and shelf-life prediction are comparable.

[0272] Example 5. Stable Liquid Formulation with Addition of Recombinant Human Serum Albumin (rHSA)

[0273] rHSA can be used in vaccine manufacturing as a supplement to ensure optimal cell growth and has been shown to be safe in multiple approved biological products (see Peters T. All About Albumin: Biochemistry, Genetics and Medical Applications. Academic Press Inc, California, USA (1996)). However, as a product excipient to stabilize enveloped virus, rHSA has not been comprehensively studied although some licensed vaccine products (i.e., MMR and Varicella) include rHSA as a residual product from the manufacturing process (see Richard T Wiedmann et al, Vaccine. 2015 Apr 27;33(18); Roman Prymul et al, BMC Pediatr. 2016; 16).

[0274] In general, there are three main modes of action for using rHSA as a stabilizer, namely, protection from sheer stress, prevention of container surface adsorption, and enhancement of overall thermal stability (see O’Neil Wiggan et al, Vaccine. 2011 October 6; 29(43)). However, no use of rHSA for stabilizing RSV liquid vaccine products has beenidentified at the time of the studies described herein. The role of rHSA in stabilization (e.g., as an excipient stabilizer) for RSV ANS2 / A1313 / 11314L was studied as described below.

[0275] 5.1 rHSA Stabilization Effect- Study 11

[0276] To better understand the role of rHSA and the minimum concentration level needed for the role of rHSA, a study was conducted with use of a non-rHSA containing RSV ANS2 / A1313 / 11314L spiked with rHSA. Two RSV ANS2 / A1313 / 11314L DS lots, a first with no rHSA (LIO) and a second spiked with 5 mg / mL rHSA (LI 1), both made from a fresh, small- scale development lot of RSV ANS2 / A1313 / 11314L DS that was never frozen). A DP formulation was made from the above DS Li l with two levels of rHSA concentrations: 0.05 mg / mL and 1.4 mg / mL. This was used to study the stabilization effect of rHSA and the impact of rHSA concentration on the stabilization effect in formulated DP.

[0277] DP after formulation in 40% trehalose, 160 mM NaCl, and 10 mM histidine at pH 7.0 samples containing 0 mg / mL rHSA, 0.05 mg / mL rHSA, and 1.4 mg / mL rHSA were compared in thermal stability at 37 °C up to 14 days.

[0278] The thermal stability and effect of rHSA effects on RSV ANS2 / A1313 / 11314L L10 and Li l, which contained either 0 or 5 mg / mL rHSA, showed that rHSA is an effective stabilizer for the DS ( - value < 0.0001).

[0279] Titer results of the DS and DP lots (see FIGs. 9A-9C) suggest that a formulation containing no rHSA exhibited significantly higher titer loss compared to DS and DP lots containing rHSA. Addition of a small amount of rHSA (at 0.05 mg / mL concentration) is shown to be effective ( -value < 0.0001). No impact was observed for freeze-thaw titer loss when the formulation contains rHSA. Therefore, the study concluded that rHSA concentrations from 0.05 mg / mL (low dose (LD) rHSA) to 1.4 mg / mL (high dose (HD) rHSA) demonstrated relatively good DP thermal stability ( / ?-values < 0.0001 vs. the control with no rHSA). rHSA below 0.05 mg / mL may result in lower stability. DS and DP with no rHSA had lower thermal stability.

[0280] 5.2 Stability Study of a Stable Formulation with Addition of DifferentConcentrations of rHSA (Study 12)

[0281] rHSA was added as stabilizer at 2 concentrations to the first 200 L RSV ANS2 / A1313 / 11314L DS lot L9 in 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0 formulation at titer target DP of 8.0 logio PFU / mL. The concentrations of rHSA were 0.05 mg / mL (F2, low concentration rHSA) and 1.4 mg / mL (F3, high concentration rHSA).

[0282] Plaque assay results at different incubation temperatures from high dose RSV ANS2 / A1313 / 11314L DP with low or high concentration rHSA are shown in FIGs. 10A-10E. The sample that was thermal stressed at 37 °C suggests that rHSA at 1.4 mg / mL can significantly reduce DP degradation compared to rHSA at 0.05 mg / mL ( / ?-value = 0.0004, see FIG. 10C).

[0283] AKTS modeling to predict the SL based on 3-month stability data is listed in Tables 13 and 14 and FIGs. 10D-10E.Table 13: AKTS SL Prediction Based on 3-Month Stability Data for F2 Formulations with 0.05 mg / mL rHSA (High Dose DP)* Estimated values based model output for information only due to insufficient dataTable 14: AKTS SL Prediction Based on 3-Month Stability Data for F3 Formulations with 1.4 mg / mL rHSA (High Dose DP)* Estimated values based model output for information only due to insufficient data

[0284] Example 6. Stable Liquid Formulation with Addition of Hydroxyethyl Starch (HES)

[0285] There were many polymers screened to examine if they could stabilize RSV live- attenuated virus, including, but not limited to, PVP, PEG, dextran, and HES. It was surprisingly observed that HES can stabilize RSV live-attenuated virus in some cases.

[0286] 6.1 HES Stabilization Effect and Concentration Study (Study 13)

[0287] It was assessed whether MSG and HES, in combination, can improve RSV thermal stability in comparison to the 40% trehalose liquid formulation (10 L DS RSV ANS2 / A1313 / 11314L L12 in 40% trehalose, 160 mM NaCl, and 10 mM histidine at pH 7.0). All samples were incubated in accelerated conditions at 37 °C for up to 14 days. For incorporating HES into the DP formulation buffer, HES powder was dissolved directly into a base DP buffer (40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7) at double the excipient target concentration. Histidine was added until the final concentration for the buffer was 30 mM, and this formulation was then mixed with the DS at a 1 : 1 ratio to achieve the final target excipient concentration. It was noted that while preparing the DP buffer with a concentration of 5% HES that HES exhibited viscous, gel-like properties in solution while it was being dissolved. After dissolution, there were no issues in passing the 5% HES DP buffer through a 0.2-pm filter unit, and there were no significant differences of formulation viscosity during the filling process.

[0288] Thermal stability of 3 formulations is summarized in FIG. 11. The study results indicated that both MSG (F2, 40% trehalose, 10 mM histidine, 160 mM NaCl, and 100 mM MSG) and HES (F3, 40% trehalose, 10 mM histidine, 160 mM NaCl + 2.5% HES, and 100 mM MSG) are significantly better than control (Fl, 40% trehalose, 10 mM histidine, 160 mM NaCl) ( - value =0.04 and - value =0.0002 up to 14 days stability at 37 °C, respectively). It is also shown that HES formulation F3 is slightly better than MSG formulation F2 when compared up to 14 days stability at 37 °C, although it is not significantly different ( - value = 0.22).

[0289] 6.2 Stability Study of a Stable Formulation with Addition of HES (Study 14)

[0290] A candidate liquid formulation for commercial use was assessed for stability performance at 3 months using AKTS modeling for shelf-life prediction. F2 is a potential formulation: 40% trehalose, 160 mM NaCl, 100 mM MSG, 20 mM histidine, and 2.5% HES at pH = 7.0. F2 was tested with RSV ANS2 / A1313 / 11314L L12

[0291] Stability samples at designated time points were assayed for infectious titer using the plaque assay described above. Raw data of this study that was used for AKTS analysis is summarized in Table 15.Table 15: Plaque Assay Titer Raw Data of F2 Formulation

[0292] One kinetic model was selected based on 6 months of data from all temperatures (5 °C, 25 °C, 37 °C, and 45 °C). FIG. 12E and Table 16 use kinetic model #56, 2-step kinetics having the best AIC and the best BIC to demonstrate the prediction models and predicted titer loss at 5 °C.Table 16 : AKTS Shelf-life Prediction Based on 24 Weeks Stability Data* Estimated values based model output for information only due to insufficient data

[0293] These studies provide data supporting use of rHSA and / or HES in combination with 40% trehalose, 160 mM NaCl, 100 mM MSG, and 20 mM histidine at pH 7.0 formulation to further improve liquid product stability.

[0294] Example 7. Other Studies

[0295] 7.1. Excipient Screening and pH Study

[0296] A study (Study 15) was conducted to confirm and optimize the 40% trehalose-based RSV ANS2 / A1313 / 11314L liquid formulations screened in 40% trehalose with 160 mM NaCl and 10 mM histidine at pH 7 with addition of screened excipients such as rHSA, PEG-400, and CaCh at different concentrations and using 10 L RSV ANS2 / A1313 / 11314L DS L13.

[0297] Impact of pH on processed 10 L scale RSV ANS2 / A1313 / 11314L DS L14 was studied (Study 16). All formulation samples were incubated at 37 °C for 1 week and 2 weeks.

[0298] Formulation compositions and thermal stability performance are summarized in Table 17.Table 17 : Summary of Studies 15 and 16

[0299] In all rHSA-containing groups (F5, F6, and F7), higher rHSA content led to a higher titer loss in 40% trehalose formulations (see Composition Concentrations in Table 17). Notably, the best formulation was F5 which was rHSA at 0.5 mg / mL. However, rHSA at 0.5 mg / mL appears to provide no significant benefit (p = 0.3275) compared to the F2 control. F8 with addition of CaCh indicated that the thermal stability performance was improved at the 1- week timepoint at 37 °C, and only 0.68 logio PFU / mL titer loss was observed. However, thermal stability performance was not improved in F9 in combination with PEG400, where the titer loss was 0.97 logio PFU / mL at the 1-week timepoint at 37 °C.

[0300] The potential stabilizers screened played a role in a lower trehalose and / or lower salt concentration formulations in other studies.

[0301] 7.2. Headspace Nitrogen Purge Study

[0302] A headspace nitrogen purge study (Study 2) was conducted to determine if nitrogen headspace could improve the stability performance of RSV ANS2 / A1313 / 11314L in different formulations. A side-by-side comparison of formulated products at selected time points is listed in Table 18 below:Table 18: Nitrogen Purge Test Plaque Assay Results

[0303] This result suggested that headspace oxygen had no effect on any of the formulations tested, suggesting that the 10 mM histidine in each of the formulations provided enough of an anti oxidation effect. 40% trehalose compared to 30% trehalose did not provide additional protection from infectious titer loss in the formulations tested. (A. Michael Wade et al., J. Nutritional Biochemistry, 1998). In another words, nitrogen headspace purge did not add benefit of preventing oxidation and improving the liquid DP in thermal stability.

[0304] Example 8. Different Drying Methods

[0305] Different drying methods were evaluated to compare the feasibility and stability of dried formulations to liquid formulations. Drying methods assessed were freeze drying, spray drying, and foam drying.

[0306] 8.1 Freeze Dry Study

[0307] Studies (Studies 1 and 18) were performed to examine the freeze-drying method on the infectious titer loss of RSV ANS2 / A1313 / 11314L in different formulations. The freeze- drying parameters used are summarized in Table 3. Formulation composition examples and their characteristics before and after drying are shown in Table 19 below. Data in Table 19 showed that the drying process loss and storage loss are very similar among three (3) formulations, in addition, three (3) formulations prepared by the freeze-drying method met the pre-defined criteria that is no more than 1 logio PFU / mL loss at a 7-day 37 °C incubation, which are considered as thermally stable dried formulations.Table 19: Titer Result Summary of Lyo Development Study 18

[0308] 8.2 Spray Dry Proof of Concept (POC) Study

[0309] A POC study (Study 19) was conducted to assess if the spray-dry method can be an effective method for stabilizing RSV ANS2 / A1313 / 11314L. The spray-dry method was assessed using 1 :6 diluted RSV ANS2 / A1313 / 11314L DS L15 (30% trehalose, 160 mM NaCl, 100 mM potassium glutamate, and 10 mM histidine at pH 7.0) and a bench spray dryer (Buchi 290). The drying parameters are listed in Table 4. Plaque assay test results conducted on predried and post-dried samples are summarized in Table 20 below:Table 20: Spray-Drying Process Loss Titer and 37 °C Storage Loss Titer Summary

[0310] Results in Table 20 suggest that the spray-dry process caused 0.64 logio PFU / mL viral titer loss, which is higher than observed in the freeze-drying process. The storage loss at 37 °C was less than 1 logio PFU / mL up to 4 weeks, which is very stable as a dried formulation.

[0311] 8.3 Foam Dry POC Study 1

[0312] A POC study (Study 20) was designed to demonstrate that a foam-dry process can be used to stabilize RSV ANS2 / A1313 / 11314L. DP was formulated using RSVANS2 / A1313 / 11314L DS L4 in 30% trehalose in 10 mM histidine, 160 mM NaCl, and 100 mM sodium glutamate at pH 7.0 and foam dried using the parameters shown in Table 5.

[0313] The stability of the foam-dried formulations was monitored at 5 ±3 °C for 12 weeks and 37 ±2 °C for 2 weeks. Infectious titer was used as a stability indicating assay in this study. The infectious titer results are summarized in FIG. 13 and Table 21.Table 21: Stability Data Summary of First Foam-Dry Study 20

[0314] This study generated 12-month data for foam-dried samples at 2-8 °C, where foamdry infectious titer loss was 0.34 logio PFU / mL at 12 months at 2-8 °C in Table 21. The drying process infectious titer loss was 0.7 logio PFU / mL.

[0315] 8.4 Foam Dry POC Study 2

[0316] Due to high drying process loss of infectious titer observed in the first study, cycle parameters were adjusted with lower temperatures for each phase of a second foam dry study to see if the infectious titer loss of RSV ANS2 / A1313 / 11314L could be decreased. The adjusted process cycle parameters are shown in Table 22. Results are shown in Table 23 and FIG. 13.

[0317] In this study, 6-month data for foam-dried samples at 2-8 °C showed foam-dry infectious titer loss is 0.37 logio PFU / mL at 6 months at 2-8 °C in Table 23. The drying process infectious titer loss was 0.5 logio PFU mL.Table 22: Second Examples of Foam-Dry Process ParametersTable 23: Foam-Dry Study 2 Plaque Assay Data

[0318] 8.5 Summary of Drying Method Comparison

[0319] Accelerated stability profiles are compared in Table 24. From this information, it was found:

[0320] (1) The second foam-drying process had a reduced titer loss of 0.52 logio PFU / mL in comparing with the first run (0.7 logio PFU / mL). It is more than freeze-drying process loss (0.3 logio PFU / mL) but slightly better than spray-dried process losses (0.6 logio PFU / mL).

[0321] (2) The second foam-dried product had more titer loss for all timepoints at 37 °C in comparison with the first run foam-dried product. The storage losses were comparable to freeze-dried and spray-dried products.

[0322] (3) The POC data suggested all drying methods had similar total loss regardless the drying process losses. The formulation is suitable for all different drying methods and yields good stable dried products.

[0323] (4) The liquid formulation demonstrated comparatively lower losses than dried formats within 2 weeks of accelerated conditions, especially for the 40% trehalose formulation, which had the best stability.

[0324] FIG. 13 summarizes the comparison of stability of different formulations and drying conditions or liquid formulations presented above.Table 24: Summary Stability Performance Comparison of Drying Methods for 30% Trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0 Formulation

[0325] The data in Table 24 provided evidence that a 30% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0 formulation with combination of salts and histidine is a stable dried viral formulation. It has also been shown that 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine at pH 7.0 formulation with combination of salts and histidine at pH 7.0 is also a stable dried viral formulation.

[0326] In summary, the RSV liquid formulation exemplified herein (i.e., having greater than 15% trehalose, a monovalent salt, and histidine) is stable as a liquid formulation for extended times at room temperature as well as in 2-8 °C. This liquid formulation can be directly administered from the shelf or 2-8 °C storage.

[0327] In comparison, US 2016 / 0250319 describes a liquid formulation as only a pre-step for bulk formulation right before drying. The liquid formulation is not suitable for direct administration to a human subject, but rather, is flash frozen to -180 °C to ice pellet before drying in order to maintain virus stability (see U

[0038] of US Patent Application Pub. No. 2016 / 0250319, which states “[i]t is preferable to freeze vaccine via flash freezing or fastfreezing approach, especially for high disaccharide containing formulationsf.]”). No normal freeze rate and F / T data was provided. Furthermore, the formulations described herein show improved stability metrics as shown in Table 25 below.Table 25: Comparison of Described Liquid Formulations (Examples in FIG. 3A, 7A, 10A, and 12A) and Dried Formulation (Examples in Table 19) to the Formulations Described in US 2016 / 0250319 at Table 5.

[0328] Stable Formulation CompositionsTable 26 : Stable Trehalose Formulation Compositions and Possible Optimal Concentrations, Optimal pH Range from 6-8.

[0329] RSV has been shown to be unstable, even in frozen conditions, and is difficult to freeze dry (Gupta CK et al., Vaccine, 1996; 14(15): 1417-20 and Liljeroos L et al., Proc Natl Acad Sci USA, 2013; 110(27): 1133-8), and therefore was an exemplary vaccine for formulation studies. In addition, live-attenuated RSV has been shown to be unstable. RSV ANS2 / A1313 / 11314L DP formulation development studies in the present disclosure covered many aspects of formulations including pH, excipients, trehalose concentration (from 15% to 40%), buffer / antioxidant histidine and monovalent salt(s), and real time storage stability. rHSA, HES, and MgCh can be added as potential stabilizers. Multiple lots of 30% trehalose formulation in both liquid and freeze-dried format were also compared. The formulations disclosed herein achieved minimal infectivity losses under stresses and demonstrated extremely low titer loss rate at 2-8 °C.

[0330] Both liquid and lyo formulations of RSV ANS2 / A1313 / 11314L in the present disclosure demonstrated suitable stability profiles in multiple lots compared to existing viral vaccine benchmarks in both accelerated and real time stability.

[0331] A stable DP formulation with 30-40% trehalose was developed. Both liquid and lyo formulations achieved excellent thermal stability performance, and performed above the other virus vaccine benchmarks when compared at 37 °C. 30-40% trehalose formulations were robust in response to many process parameters such as dilution, mixing shear stress, headspace, etc. Furthermore, 30-40% trehalose formulations can be flexible as frozen liquid, liquid, and freeze-dried formulations based on the stage and needs of early and late development phases.

[0332] Study results also showed that 40% trehalose formulation is a stable liquid formulation for the RSV ANS2 / A1313 / 11314L live-attenuated virus. In general, 40% trehaloseliquid formulation provides at least 12-24 months of stability. Three potential stabilizers MgCh, rHSA, and HES demonstrated evidence of stabilizing RSV ANS2 / A1313 / 11314L.

[0333] Example 9. HSV-2 Trehalose FormulationTo investigate the effect of trehalose and MSG on stabilization of HSV-2 live-attenuated virus (HSV 529 as described in Da Costa, X.; Kramer, M.F.; Zhu, J.; Brockman, M.A.; Knipe, D.M. Construction, phenotypic analysis, and immunogenicity of a UL5 / UL29 double deletion mutant of herpes simplex virus 2. J. Virol., 2000, 74, 7963-7971), a full factorial study was performed on lyophilized process. The study design had two variables with three levels: trehalose dehydrate at 10%, 20%, and 30% and MSG at 0 mM, 50 mM, and 100 mM. The result is summarized in the Table 27 below. All formulations were compared to the control 10% sucrose, 160 mM NaCl, 50 mM MSG, and 10 mM histidine buffer at pH 7.0.Table 27: Loss Effect of Trehalose and MSG on Titer Loss During Lyophilization and Storage

[0334] The results (Table 27) from this study indicated that increasing the concentration of trehalose can significantly reduce lyophilization loss. Interestingly, the formulation containing 30% trehalose with 100 mM MSG was the only formulation that showed significantly better results when compared to control and met the stability criteria. Although trehalose was the only ingredient that significantly impacted the lyophilization loss, there was a clear interaction with MSG levels.

[0335] A confirmation stability study done using HSV-2 DS at a titer of 7.99 logio PFU / mL in HSV-2 lyo buffer (30% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine pH 7.0) was directly filled into 0.5 mL to 3 mL glass vials. The lyo cycle used was the same as parameters in the optimized process of Table 3.

[0336] The result of this confirmation run (Table 28, FIG. 14) suggested that 30% trehalose formulation greatly stabilized HSV-2 with a lyo presentation. The titer loss was 0.32 logio PFU / mL for lyo process; the titer loss was 1 logio PFU / mL when stored at 37 °C, 4 week time point, respectively. However, it was not able to stabilize HSV-2 in a liquid presentation at all as titer was completely lost when stored at 25 °C for 1 week.Table 28: Summary of Infectious Titer for HSV-230% Trehalose Formulation

[0337] Example 10. Yellow Fever Trehalose Formulation

[0338] Data was also generated to assess the formulations described herein and tested on RSV for applicability to other live-attenuated viruses, e.g., a yellow fever live-attenuated virus. The stability data is summarized in Table 29 below.Table 29: Yellow Fever Lyophilized Product Stability in 30% Trehalose Formulation

[0339] In comparison, the 30% trehalose formulation (YF F2: trehalose 331.6 mg / mL; NaCl 9.35 mg / mL; MSG 18.713 mg / mL; histidine 9.35 mg / mL pH 7.0) had slightly better stabilization performance than the original yellow fever formulation (YF FL trehalose 150 mg / mL; PVP10 20 mg / mL; CaCh. H2O 1.325 mg / mL; Proline 3.2 mg / mL; Urea 2.5 mg / mL; P407 5 mg / mL; Lysine. H2O 0.337mg / mL; Sorbitol 30 mg / mL in 20 mM Tris buffer pH 8.0) in both lyo loss and storage loss, respectively. 30% trehalose formulations were able to stabilize the yellow fever virus up to 2 weeks at 37 °C incubation with about 1.06 logio Cell Culture Infectious Dose, 50% endpoint (CCID50) titer loss.

[0340] Example 11. PIV Trehalose Formulation

[0341] Liquid and lyophilized vector-based formulations comprising PIV are prepared according to the methods described herein and tested for stability at 2-8 degrees Celsius, room temperature, and 37 degrees Celsius. A PIV3 vector-based vaccine is prepared comprising a PIV3 derived vector virus and one or more heterologous RSV or hMPV antigens. In some embodiments, the RSV antigen is RSV F and / or RSV G. A PIV3 vector-based vaccine is also prepared comprising a PIV3 derived vector virus and a non-PIV heterologous antigen.

[0342] Trehalose concentrations of 15% and greater (e.g., 30% and 40% trehalose) provide stabilization to liquid formulations comprising PIV similar to the RSV and HSV-2 based formulations described above.APPENDIX A: SEQUENCE SUMMARYDESCRIPTION OF THE SEQUENCES

[0343] Appendix A provides a listing of certain sequences referenced herein. The amino acid sequences provided are from N-terminus to C-terminus. The nucleic acid sequences are from 5’ to 3’.

Claims

What is claimed is:

1. A liquid, lyophilized, or frozen formulation comprising: a. an effective amount of live-attenuated and / or vector-based virus; b. about 30 to about 45% (w / v) trehalose; c. one or more monovalent salts; and d. a buffer and / or antioxidant comprising histidine.

2. A liquid, lyophilized, or frozen formulation comprising an effective amount of live- attenuated and / or vector-based virus and from about 30% to about 40% (w / v) trehalose, about 100 mM monosodium glutamate (MSG), about 160 mM NaCl, and about 10 mM histidine.

3. A liquid, lyophilized, or frozen formulation comprising: a. an effective amount of live-attenuated respiratory syncytial virus (RSV); b. about 30 to about 45% (w / v) trehalose; c. one or more monovalent salts; and d. a buffer and / or antioxidant comprising histidine.

4. A liquid, lyophilized, or frozen formulation comprising: a. an effective amount of live-attenuated RSV; b. about 30 to about 45 % (w / v) trehalose; c. about 10 to about 300 mM NaCl; d. about 0.5 to about 300 mM monosodium glutamate (MSG) or potassium glutamate; and e. about 10 to about 100 mM histidine; wherein the formulation has a pH of about 6 to about 8.

5. The formulation of claim 1 or claim 2, wherein the virus is enveloped.

6. The formulation of any one of claims 1-4, wherein the virus is RSV ANS2 / A1313 / 11314L.

7. The formulation of claim 6, wherein the effective amount of the RSVANS2 / A1313 / 11314L is about 5 to about 9 logio plaque forming units (PFU) per dose.

8. The formulation of claim 6, wherein the effective amount of the RSVANS2 / A1313 / 11314L is about 5 to about 6 logio PFU per dose or about 6.1 to about 7 logio PFU per dose or about 7.1 to about 8 logio PFU per dose or about 8.1 to about 9 logio PFU per dose.

9. The formulation of claim 6, wherein the effective amount of the RSV ANS2 / A1313 / 11314L is about 5.6 logio PFU per dose or about 6.2 logio PFU per dose.

10. The formulation of any one of claims 3 or 4, wherein a codon in the live-attenuated RSV that encodes a serine at position 1313 of the L protein is deleted resulting in the deletion of the amino acid in the L protein (A1313).

11. The formulation of any one of claims 3, 4, or 10, wherein an amino acid residue substitution of leucine for isoleucine at position 1314 with reference to SEQ ID NO: 3 in the RSV results in a genetically stabilizing mutation in the L gene (11314L).

12. The formulation of any one of claims 3, 4, or 10-11, wherein the RSV comprises: a large polymerase protein (L), a phosphoprotein (P), a nucleocapsid protein (N), an M2-1 protein nonstructural protein 1 (NS1), a glycoprotein (G), a fusion protein (F), a matrix protein (M), an M2-2 protein, and a small hydrophobic protein (SH); and a genome or antigenome comprising a deletion of the codon that encodes the serine at position 1313, or a corresponding position, of the L protein; a mutation of amino acid sequence residue 1314, or a corresponding position, of the L protein, wherein the mutation of L protein amino acid sequence residue 1314 is an amino acid substitution of leucine for isoleucine, wherein the leucine is encoded by a codon set forth as CTG; a deletion of the NS2 gene; and a nucleotide modification at position 14456 with reference to SEQ ID NO: 1 that represents a change from a thymine (T) to an adenine (A).

13. The formulation of any one of the previous claims, comprising a liquid formulation.

14. The formulation of any one of the previous claims, comprising a lyophilized formulation.

15. The formulation of claim 13, wherein the liquid formulation is formulated for administration to a human subject.

16. The formulation of claim 15, wherein the administration is intranasal.

17. The formulation of any one of the preceding claims, wherein the formulation does not comprise a polyoxyethylene-polyoxypropylene (PEO-PPO) block copolymer.

18. The formulation of any one of the preceding claims, which is a liquid formulation, wherein the liquid formulation is not subsequently dried and reconstituted.

19. The formulation of any one of claims 1 and 3, which further comprises monosodium glutamate (MSG) or potassium glutamate (PG).

20. The formulation of any one of claims 1 and 3, comprising about 10 to about 100 mM histidine.

21. The formulation of any one of the preceding claims, which comprises about 30% trehalose.

22. The formulation of any one of the preceding claims, which comprises about 40% trehalose.

23. The formulation of any one of the preceding claims, which comprises about 0.5 to about 300 mM monosodium glutamate (MSG) or potassium glutamate (PG).

24. The formulation of any one of claims 1 or 3, wherein the monovalent salt is NaCl.

25. The formulation of any one of claims 1, 3, 5, or 20-22, which comprises about 10 to about 300 mM NaCl.

26. The formulation of any one of the preceding claims, wherein the pH is about 6 to about 8.

27. The formulation of any one of the preceding claims, wherein the pH is about 7 ± 0.5.

28. The formulation of any one of claims 1 or 2, wherein the live-attenuated virus comprises a paramyxovirus.

29. The formulation of any one of claims 1, 2, 5, or 28, wherein the live-attenuated virus comprises a respiratory syncytial virus (RSV).

30. The formulation of any one of the preceding claims, wherein the virus is RSV, and wherein the RSV is for administration to pediatric subjects.

31. The formulation of any one of claims 1, 2, 5, or 20-22, comprising a vector-based PIV virus, optionally wherein the PIV is PIV3.

32. The formulation of claim 31, wherein the PIV comprises one or more heterologous RSV or hMPV antigens.

33. The formulation of claim 32, wherein the RSV antigen is RSV F and / or RSV G.

34. The formulation of any one of the preceding claims, wherein the formulation is liquid, and wherein there is a titer loss of less than 1 logio PFU / mL when stored at about 2 to about 8 degrees Celsius for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months.

35. The formulation of claim 34, wherein there is a titer loss of less than 1 logio PFU / mL after about 12 to about 24 months in storage at about 2 to about 8 degrees Celsius.

36. The formulation of any one of the preceding claims, wherein the formulation is liquid, and wherein there is a titer loss of less than 1 logio PFU / mL when stored at about 37 degrees Celsius for at least 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days.

37. The formulation of claim 34, wherein there is a titer loss of less than 1 logio PFU / mL for about 1 to about 2 weeks at 37 °C.

38. The formulation of any one of the preceding claims, wherein the formulation is liquid, and wherein there is a titer loss of less than 1 logio PFU / mL when stored at room temperature for at least 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, or 5-6 months.

39. The formulation of claim 38, wherein there is a titer loss of less than 1 logio PFU / mL at room temperature for 3-4 months.

40. The formulation of any one of claims 1-33, wherein the formulation is lyophilized, and wherein there is a titer loss of less than 1 logio PFU / mL for about 1 to about 2 weeks at 37 °C.

41. The formulation of any one of claims 1-33, wherein the formulation is lyophilized, and wherein there is a titer loss of less than 1 logio PFU / mL when stored at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.

42. The formulation of any one of the preceding claims, wherein the infectious titer is not substantially lost upon shear stress, multiple freeze thaws, or reconstitution.

43. The formulation of any one of the preceding claims, wherein the vaccine is formulated to be administered intramuscularly or subcutaneously.

44. A formulation as claimed in any one of the preceding claims, which additionally comprises an adjuvant.

45. A formulation as claimed in any one of the preceding claims, which additionally comprises rHSA, a bivalent salt, and / or amino acids.

46. A method of increasing the stability of a liquid or lyophilized vaccine at room temperature or at a temperature of about 2 to about 8 degrees Celsius comprising formulating a live-attenuated enveloped virus and / or vector-based virus in a liquid or lyophilized formulation comprising about 30 to about 45% (w / v) trehalose, one or more monovalent salts, and a buffer and / or antioxidant comprising histidine.

47. The method of claim 46, wherein the concentration of trehalose is about 30% (w / v).

48. The method of claim 46, wherein the concentration of trehalose is about 40% (w / v).

49. A method of immunizing a subject against a viral infection comprising administering the formulation of any one of claims 1-48.

50. A liquid, lyophilized, or frozen formulation comprising: a. an effective amount of live-attenuated RSV comprising RSV ANS2 / A1313 / 11314L; b. about 30 to about 40 % (w / v) trehalose;c. about 160 mM NaCl; d. about 100 mM monosodium glutamate (MSG); and e. about 10 mM histidine; wherein the formulation has a pH of about 7.

51. The formulation of claim 50, wherein the formulation comprises about 30 % (w / v) trehalose.

52. The formulation of claim 50, wherein the formulation comprises about 40 % (w / v) trehalose.

53. The formulation of any one of claims 50-52, wherein the live-attenuated RSV comprises: a large polymerase protein (L), a phosphoprotein (P), a nucleocapsid protein (N), an M2-1 protein nonstructural protein 1 (NS1), a glycoprotein (G), a fusion protein (F), a matrix protein (M), an M2-2 protein, and a small hydrophobic protein (SH); and a genome or antigenome comprising a deletion of the codon that encodes the serine at position 1313, or a corresponding position, of the L protein; a mutation of amino acid sequence residue 1314, or a corresponding position, of the L protein, wherein the mutation of L protein amino acid sequence residue 1314 is an amino acid substitution of leucine for isoleucine, wherein the leucine is encoded by a codon set forth as CTG; a deletion of the NS2 gene; and a nucleotide modification at position 14456 with reference to SEQ ID NO: 1 that represents a change from a thymine (T) to an adenine (A).

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